Photoactive indicator compounds

ABSTRACT

Photoactive indicator compounds of the formula ##STR1##

This is a divisional of application Ser. No. 08/117,365, filed Sep. 3,1993 now abandoned.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to methods, compositions and kits for determiningan analyte in a sample. In particular, this invention relates tospecific binding assays which utilize a photoactive indicator precursorwhich can react with singlet oxygen to form a fluorescent product.

The clinical diagnostic field has seen a broad expansion in recentyears, both as to the variety of materials (analytes) that may bereadily and accurately determined, as well as the methods for thedetermination. Convenient, reliable and non-hazardous means fordetecting the presence of low concentrations of materials in liquids isdesired. In clinical chemistry these materials may be present in bodyfluids in concentrations below 10⁻¹² molar. The difficulty of detectinglow concentrations of these materials is enhanced by the relativelysmall sample sizes that can be utilized.

In developing an assay there are many considerations. One considerationis the signal response to changes in the concentration of an analyte. Asecond consideration is the ease with which the protocol for the assaymay be carried out. A third consideration is the variation ininterference from sample to sample. Ease of preparation and purificationof the reagents, availability of equipment, ease of automation andinteraction with material of interest are some of the additionalconsiderations in developing a useful assay.

One broad category of techniques involves the use of a receptor whichcan specifically bind to a particular spacial and polar organization ofa labeled ligand as a function of the presence of an analyte. Theobserved effect of binding by the receptor will depend upon the label.In some instances the binding of the receptor merely provides for adifferentiation in molecular weight between bound and unbound labeledligand. In other instances the binding of the receptor will facilitateseparation of bound labeled ligand from free labeled ligand or it mayaffect the nature of the signal obtained from the label so that thesignal varies with the amount of receptor bound to labeled ligand. Afurther variation is that the receptor is labeled and the ligandunlabeled. Alternatively, both the receptor and ligand are labeled ordifferent receptors are labeled with two different labels, whereupon thelabels interact when in close proximity and the amount of ligand presentaffects the degree to which the labels of the receptor may interact.

There is a continuing need for new and accurate techniques that can beadapted for a wide spectrum of different ligands or be used in specificcases where other methods may not be readily adaptable.

Homogeneous immunoassays in which it is unnecessary to separate thebound and unbound label have previously been described for smallmolecules. These assays include SYVA's FRAT® assay, EMIT® assay, enzymechanneling immunoassay, and fluorescence energy transfer immunoassay(FETI); enzyme inhibitor immunoassays (Hoffmann LaRoche and AbbottLaboratories): fluorescence polarization immunoassay (Dandlicker), amongothers. All of these methods have limited sensitivity, and only a fewincluding FETI and enzyme channeling, are suitable for largemultiepitopic analytes.

Hererogenous immunoassays in which a separation step is required aregenerally useful for both small and large molecules. Various labels havebeen used including enzymes (ELISA), fluorescent labels (FIA),radiolabels (RIA), chemiluminescent labels (CLA), etc.

Luminescent compounds, such as fluorescent compounds andchemiluminescent compounds, find wide application in the assay fieldbecause of their ability to emit light. For this reason, luminescershave been utilized as labels in assays such as nucleic acid assays andimmunoassays. For example, a member of a specific binding pair isconjugated to a luminescer and various protocols are employed. Theluminescer conjugate can be partitioned between a solid phase and aliquid phase in relation to the amount of analyte in a sample suspectedof containing the analyte. By measuring the luminescence of either ofthe phases, one can relate the level of luminescence observed to aconcentration of the analyte in the sample.

Particles, such as latex beads and liposomes, have also been utilized inassays. For example, in homogeneous assays an enzyme may be entrapped inthe aqueous phase of a liposome labelled with an antibody or antigen.The liposomes are caused to release the enzyme in the presence of asample and complement. Antibody- or antigen-labelled liposomes, havingwater soluble fluorescent or non-fluorescent dyes encapsulated within anaqueous phase or lipid soluble dyes dissolved in the lipid bilayer ofthe lipid vesicle, have also been utilized to assay for analytes capableof entering into an immunochemical reaction with the surface boundantibody or antigen. Detergents have been used to release the dyes fromthe aqueous phase of the liposomes. Particles have been dyed withfluorescent dyes and used as labels in immunoassays. Undyed particleshave also been used (e.g., latex agglutination).

Related Art

European Published Patent Application No. 0 345 776 (McCapra) disclosesspecific binding assays that utilize a sensitizer as a label. Thesensitizers include any moiety which, when stimulated by excitation withradiation of one or more wavelengths or other chemical or physicalstimulus (e.g., electron transfer, electrolysis, electroluminescence orenergy transfer) will achieve an excited state which (a) uponinteraction with molecular oxygen will produce singlet molecular oxygen,or (b) upon interaction with a leuco dye will assume a reduced form thatcan be returned to its original unexcited state by interaction withmolecular oxygen resulting in the production of hydrogen peroxide.Either interaction with the excited sensitizer will, with the additionof reagents, produce a detectible signal.

European Published Patent Application No. 0 476 556 (Motsenbocker)discloses a method for determination of a light sensitive substancewherein irradiation of lumigenic substance-light sensitive substancesolution with modulated light is used to generate short wavelength lightproportionally to the concentration of the light sensitive substance.

Luminescent labels for immunoassays are described in McCapra et al.,Journal of Bioluminescence and Chemiluminescence (1989), Vol. 4, pp.51-58.

European Published Patent Application No. 0 515 194 (Ullman et al.)discloses methods for determining an analyte in a medium suspected ofcontaining the analyte. One such disclosed method comprises treating amedium suspected of containing an analyte under conditions such that theanalyte, if present, causes a photosensitizer and a chemiluminescentcompound to come into close proximity. The photosensitizer generatessinglet oxygen and activates the chemiluminescent compound when it is inclose proximity. The activated chemiluminescent subsequently produceslight upon activation by singlet oxygen. The amount of light produced isrelated to the amount of analyte in the medium.

In this method, each singlet oxygen that is generated can react with nomore than one chemiluminescent compound, which, in turn, can emit notmore than one photon of light. The sensitivity of the method istherefore limited by the chemiluminescence quantum efficiency of thechemiluminescent compound, and, more importantly, by the ability todetect the limited number of photons that will be emitted upon reactionwith singlet oxygen.

SUMMARY OF THE INVENTION

The present invention is directed to methods for determining an analyte,kits for conducting assays for an analyte, and compounds useful in themethods and assays.

One aspect of the invention is a method for determining an analyte whichis an specific binding pair (sbp) member. In one embodiment of thisaspect the method comprises a first step of providing in combination amedium suspected of containing an analyte; a photosensitizer capable inits excited state of generating singlet oxygen, wherein thephotosensitizer is associated with a sbp member; and a photoactiveindicator precursor capable of forming a photoactive indicator uponreaction with singlet oxygen, wherein the photoactive indicatorprecursor is associated with an sbp member; then a second step ofexciting the photosensitizer by irradiation with light; and a final stepof measuring the fluorescence of the photoactive indicator. At least oneof the sbp members is capable of binding directly or indirectly to theanalyte or to an sbp member complementary to the analyte. Thefluorescence measured is related to the amount of the analyte in themedium.

In another embodiment, the method comprises the first step of combiningin an aqueous medium a sample suspected of containing an analyte; afirst suspendible particle comprised of a photosensitizer capable in itsexcited state of generating singlet oxygen, wherein the particle has aspecific binding pair (sbp) member bound thereto; and a secondsuspendible particle comprised of a photoactive indicator precursorcapable of forming a photoactive indicator upon reaction with singletoxygen, wherein the particle has a sbp member bound thereto; a secondstep of irradiating the medium to excite the photosensitizer to generatesinglet oxygen; and a final step of measuring the fluorescence of thephotoactive indicator. Each sbp member is capable of binding directly orindirectly with the analyte or to an sbp member complementary to theanalyte. The fluorescence measured is related to the amount of theanalyte in the medium.

In another embodiment, the method comprises a first step of providing incombination a medium suspected of containing an analyte; aphotosensitizer capable in its excited state of generating singletoxygen, wherein the photosensitizer is associated with a sbp member; anda suspendible particle having bound thereto an sbp member, wherein thesuspendible particle comprises a photoactive indicator precursor capableof forming a photoactive indicator upon reaction with singlet oxygen; asecond step of irradiating the combination with light to excite thephotosensitizer; and a final step of measuring the fluorescence of thephotoactive indicator. Each sbp member is capable of binding directly orindirectly to the analyte or to a sbp member complementary to theanalyte. The fluorescence measured is related to the amount of theanalyte in the medium.

Another aspect of the invention is a method for determining an analyte.The method comprises a first step of providing in combination a mediumsuspected of containing an analyte; a photosensitizer capable in itsexcited state of generating singlet oxygen, wherein the photosensitizeris associated with a first specific binding pair (sbp) member; and aphotoactive indicator precursor capable of forming a photoactiveindicator upon reaction with singlet oxygen, wherein the photoactiveindicator precursor is associated with a second sbp member; a secondstep of irradiating the combination with light to excite thephotosensitizer; and a final step of measuring the fluorescence of thephotoactive indicator. Each sbp member is capable of binding directly orindirectly to the analyte or to a sbp member complementary to theanalyte. The fluorescence measured is related to the amount of theanalyte in the medium.

Another aspect of this invention is a method for determining apolynucleotide analyte. The method comprises a first step of combiningin an aqueous medium the analyte; one or more polynucleotide probes(wherein each probe contains a nucleotide sequence complementary to anucleotide sequence of the analyte and wherein at least one probe isassociated with a specific binding pair (sbp) member that is differenctfrom said complementary nucleotide sequence); a photosensitizer capablein its excited sate of generating singlet oxygen (wherein saidphotosensitizer is associated with a polynucleotide having a sequencecomplementary to a nucleotide sequence of said probe); and a photoactiveindicator precursor capable of forming a photoactive indicator uponreaction with singlet oxygen, wherein the photoactive indicatorprecursor is associated with an sbp member complementary to the sbpmember associated with the probe; a second step of irradiating themedium with light to excite the photosensitizer to generate singletoxygen; and a third step of measuring the fluorescence of thephotoactive indicator. The fluorescence is related to the amount of theanalyte in the medium.

Another aspect of this invention is a composition comprising suspendibleparticles of average diameter of 20 to 4000 nanometers having associatedtherewith a photoactive indicator precursor, wherein the photoactiveindicator precursor contains an selenium or tellurium atom.

Another aspect of this invention is a kit for conducting an assay foranalyte. The kit comprises, in packaged combination, suspendibleparticles comprising a photoactive indicator precursor, wherein saidphotoactive indicator precursor contains a selenium or a tellurium atomand wherein the particles have bound thereto a sbp member; and aphotosensitizer which is associated with a sbp member and is capable inits excited state of activating oxygen to its singlet state, wherein atleast one of the sbp members is capable of binding to the analyte or toan sbp member complementary to the analyte.

In another embodiment of this aspect, the kit comprises, in packagedcombination, a composition, which comprises a first suspendible particlecomprising a photoactive indicator precursor containing a selenium ortellurium atom, wherein the first particle has bound thereto a sbpmember; and a second suspendible particle comprising a photosensitizer,wherein the second particle has bound thereto a sbp member. At least oneof the sbp members is capable of binding to the analyte or to an sbpmember complementary to the analyte.

In another embodiment of this aspect, the kit comprises, in packagedcombination, a photoactive indicator precursor containing a selenium ortellurium atom, wherein the photoactive indicator precursor isassociated with a first sbp member; and a photosensitizer capable in itsexcited state of activating oxygen to its singlet state associated witha second sbp member. The sbp members are capable of binding to theanalyte or to a sbp member capable of binding the analyte.

Another aspect of this invention is a binding assay for an analyte thatis a sbp member. The assay comprises the first step of combining amedium suspected of containing the analyte with a sbp member capable ofbinding directly or indirectly to the analyte or to a sbp membercomplementary to the analyte; a second step of detecting the binding ofthe sbp member to the analyte or the complementary sbp member, whereinthe detection comprises exposing a photoactive indicator precursor inthe medium to singlet oxygen to produce a photoactive indicator; and afinal step of measuring the fluorescence of the photoactive indicator.

Another aspect of this invention are compounds useful as photoactiveindicator precursors which contain the following structure: ##STR2##wherein H is cis to the XR group; X is a selenium or tellurium; R is anorganic or organometallic group bound to X through an unsaturated carbonatom, a silicon atom, or a tin atom; and A, when taken with thecarbon-carbon group, forms an alicyclic ring (optionally fused to one ormore aromatic rings) or a heterocyclic ring; where upon reaction of thecompound with singlet oxygen, the H and the XR group are replaced by acarbon-carbon double bond to yield a fluorescent molecule having anextinction coefficient of at least 10,000 M⁻¹ cm⁻¹ at its absorptionmaximum and a fluorescence emission quantum yield of at least 0.1.

Another aspect of this invention is a method for preparing a photoactiveindicator molecule. The method comprises reacting a compound of theinvention (as described above) with singlet oxygen to yield aphotoactive indicator having an extinction coefficient of at least10,000 M⁻¹ cm⁻¹ at its absorption maximum and a fluorescence emissionquantum yield of at least 0.1.

One of the advantages of the present invention is the ability of thefluorescent photoactive indicator (which is produced from the reactionof the photoactive indicator precursor with singlet oxygen) to generateat least 10⁻⁵ times as many photons as the chemiluminescent compoundused in the method described above in European Published PatentApplication No. 0 515 194. This is because a single fluorescentphotoactive indicator molecule can often be excited up to 10⁻⁵ timesbefore it is destroyed. Thus, the fluorescent photoactive indicatormolecule that is formed in the present invention can produce tens ofthousands of photons on irradiation. Detection of this fluorescence cantherefore provide a more sensitive assay. Moreover, measurement of thefluorescence of the photoactive indicator molecule in the presentinvention permits the use of a standard fluorometer whereas detection ofthe chemiluminescence produced on activation of the chemiluminescentcompound in the previously described assay requires more specializedspectrometers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic depiction of the results of DNA detection assays.The results of each assay are depicted by a different symbol.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in this specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated:

"Alkyl" refers to a monovalent branched or unbranched radical derivedfrom an aliphatic hydrocarbon by removal of one hydrogen atom; includesboth lower alkyl and upper alkyl.

"Lower alkyl" refers to an alkyl radical containing from 1 to 5 carbonatoms, e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl,isopentyl, and the like.

"Upper alkyl" refers to an alkyl radical containing more than 6 carbonatoms, usually 6 to 20 carbon atoms, e.g., hexyl, heptyl, octyl, and thelike.

"Alkylidene" refers to a divalent organic radical derived from an alkylradical in which two hydrogen atoms are taken from the same carbon atom,e.g., ethylidene, and the like.

"Alkylene" refers to a divalent organic radical derived from an alkylradical in which two hydrogen atoms are taken from different carbonatoms.

"Alicyclic ring" refers to a cyclic hydrocarbon radical of 5 to 7carbons in length which may be unsaturated or partially saturated.

"Aryl" refers to an organic radical derived from an aromatic hydrocarbonby the removal of one atom and containing one or more aromatic rings,usually one to four aromatic rings, e.g., phenyl (from benzene),naphthyl (from naphthalene), and the like.

"Aralkyl" refers to an organic radical having an alkyl group to which isattached an aryl group, e.g., benzyl, phenethyl, 3-phenylpropyl,1-naphthylethyl, and the like.

"Alkoxy" refers to a radical of the formula --OR_(a) where R_(a) is analkyl group, e.g., methoxy, ethoxy, and the like.

"Aryloxy" refers to a radical of the formula --OR_(b) where R_(b) is anaryl group, e.g., phenoxy, naphthoxy, and the like.

"Aralkoxy" refers to a radical of the formula --OR_(c) where R_(c) is anaralkyl radical, e.g., benzyloxy, 1-naphthylethoxy, and the like.

"Alkylthio" refers to a radical of the formula --SR_(a) where R_(a) isan alkyl group, e.g., methylthio, ethylthio, and the like.

"Arylthio" refers to a radical of the formula --SR_(b) where R_(b) is anaryl group, e.g., phenylthio, naphthylthio, and the like.

"Heterocyclic ring" refers to a stable mono-, bi- or tricyclic ringsystem which consists of carbon atoms and from one to three heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur andwhich is either saturated or unsaturated, wherein the nitrogen, carbonor sulfur atoms may optionally be oxidized, and the nitrogen atom mayoptionally be quaternized, and includes any ring system in which any ofthe above-defined heterocyclic ring systems is fused to a benzene ring.The heterocyclic ring system may be substituted at any heteroatom orcarbon atom which results in the creation of a stable structure.Examples of such heterocyclic ring systems include, but are not limitedto, piperidine, piperazine, 2-oxopiperazine, 2-oxopiperidine,2-oxopyrrolidine, 2-oxoazepine, azepine, pyrrole, 4-piperidone,pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, pyrazine, pyrimidine, pyridazine, oxazole,oxazolidine, indane, isoxazole, isoxazolidine, morpholine, thiazole,thiazolidine, isothiazole, quinuclidine, isothiazolidine, indole,isoindole, indoline, isoindoline, octahydroindole, octahydroisoindole,quinoline, isoquinoline, decahydroisoquinoline, benzimidazole,thiadiazole, dihydrobenzofuran, benzofuran, benzopyran, 1,4-benzopyrone,1,2-benzopyrone, benzothiazole, benzoxazole, furan, tetrahydrofuran,pyran, tetrahydropyran, thiophene, benzothiophene, thiamorpholine,thiamorpholine sulfoxide, thiamorpholine sulfone, and oxadiazole.Preferred heterocyclic rings for the purposes of this invention arebenzopyrones.

"Substituted" refers to the condition wherein a hydrogen atom of amolecule has been replaced by another atom, which may be a single atomsuch as a halogen, etc., or part of a group of atoms, such as an organicgroup.

"Electron-donating group" refers to a substituent which, when bound to amolecule, is capable of polarizing the molecule such that theelectron-donating group becomes electron poor and positively hargedrelative to another portion of the molecule, i.e., has reduced electrondensity. Such groups may be, by way of illustration and not limitation,amines, ethers, thioethers, phosphines, hydroxy, oxyanions, mercaptansand their anions, sulfides, etc.

"Organic group" refers to a substituent having from 1 to 50 atoms otherthan the requisite number of hydrogen atoms necessary to satisfy thevalencies of the atoms in the radical. Generally, the predominant atomin such a group is carbon (C) but may also be oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), wherein, if present, the O, N, S, or P atommay be bound to carbon or to one or more of each other or to hydrogen orto a metal atom to form various functional groups, such as carboxylicacids, alcohols, thiols, carboxamides, carbamates, carboxylic acidesters, sulfonic acids, sulfonic acid esters, phosphoric acids,phosphoric acid esters, ureas, carbamates, phosphoramides, sulfonamides,ethers, sulfides, thioethers, olefins, acetylenes, amines, ketones,aldehydes, nitriles, and the like. Illustrative of such organic groups,by way of illustration and not limitation, are alkyl, alkylidine, aryl,aralkyl, and heterocyclyl, wherein the alkyl, alkylidine, aryl, aralkylor heterocyclyl group may be substituted with one or more of theaforementioned functional groups.

"Organometallic group" refers to a radical containing an organic group(as defined above) linked to a metal atom.

"Analyte" refers to the compound or composition to be detected. Theanalyte can be comprised of a member of a specific binding pair (sbp)and may be a ligand, which is monovalent (monoepitopic) or polyvalent(polyepitopic), usually antigenic or haptenic, and is a single compoundor plurality of compounds which share at least one common epitopic ordeterminant site. The analyte can be a part of a cell such as bacteriaor a cell bearing a blood group antiget such as A, B, D, etc., or an HLAantigen or a microorganism, e.g., bacterium, fungus, protozoan, orvirus.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes to which the subjectinvention can be applied will have a molecular weight of at least about5,000, more usually at least about 10,000. In the poly(amino acid)category, the poly(amino acids) of interest will generally be from about5,000 to 5,000,000 molecular weight, more usually from about 20,000 to1,000,000 molecular weight; among the hormones of interest, themolecular weights will usually range from about 5,000 to 60,000molecular weight.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immunoglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc.

The types of proteins, blood clotting factors, protein hormones,antigenic polysaccharides, microorganisms and other pathogens ofinterest in the present invention are specifically disclosed in U.S.Pat. No. 4,650,770, the disclosure of which is incorporated by referenceherein in its entirety.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.

The analytes include drugs, metabolites, pesticides, pollutants, and thelike. Included among drugs of interest are the alkaloids. Among thealkaloids are morphine alkaloids, which includes morphine, codeine,heroin, dextromethorphan, their derivatives and metabolites; cocainealkaloids, which include cocaine and benzyl ecgonine, their derivativesand metabolites; ergot alkaloids, which include the diethylamide oflysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazolinealkaloids; isoquinoline alkaloids; quinoline alkaloids, which includequinine and quinidine; diterpene alkaloids, their derivatives andmetabolites.

The next group of drugs includes steroids, which includes the estrogens,androgens, andreocortical steroids, bile acids, cardiotonic glycosidesand aglycones, which includes digoxin and digoxigenin, saponins andsapogenins, their derivatives and metabolites. Also included are thesteroid mimetic substances, such as diethylstilbestrol.

The next group of drugs is lactams having from 5 to 6 annular members,which include the barbiturates, e.g., phenobarbital and secobarbital,diphenylhydantoin, primidone, ethosuximide, and their metabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes the amphetamines; catecholamines, whichincludes ephedrine, L-dopa, epinephrine; narceine; papaverine; andmetabolites of the above.

The next group of drugs is benzheterocyclics which include oxazepam,chlorpromazine, tegretol, their derivatives and metabolites, theheterocyclic rings being azepines, diazepines and phenothiazines.

The next group of drugs is purines, which includes theophylline,caffeine, their metabolites and derivatives.

The next group of drugs includes those derived from marijuana, whichincludes cannabinol and tetrahydrocannabinol.

The next group of drugs is the hormones such as thyroxine, cortisol,triiodothyronine, testosterone, estradiol, estrone, progesterone,polypeptides such as angiotensin, LHRH, and immunosuppressants such ascyclosporin, FK506, mycophenolic acid, and so forth.

The next group of drugs includes the vitamins such as A, B (e.g., B₁₂),C, D, E and K, folic acid, thiamine.

The next group of drugs is prostaglandins, which differ by the degreeand sites of hydroxylation and unsaturation.

The next group of drugs is the tricyclic antidepressants, which includeimipramine, dismethylimipramine, amitriptyline, nortriptyline,protriptyline, trimipramine, chlomipramine, doxepine, anddesmethyldoxepin,

The next group of drugs are the anti-neoplastics, which includemethotrexate.

The next group of drugs is antibiotics, which include penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, the metabolitesand derivatives.

The next group of drugs is the nucleosides and nucleotides, whichinclude ATP, AND, FMN, adenosine, guanosine, thymidine, and cytidinewith their appropriate sugar and phosphate substituents.

The next group of drugs is miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, lidocaine, procainamide,acetylprocainamide, propranolol, griseofulvin, valproic acid,butyrophenones, antihistamines, chloramphenicol, anticholinergic drugs,such as atropine, their metabolites and derivatives.

Metabolites related to diseased states include spermine, galactose,phenylpyruvic acid, and porphyrin Type 1.

The next group of drugs is aminoglycosides, such as gentamicin,kanamycin, tobramycin, and amikacin.

Among pesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sullenamides, theirmetabolites and derivatives.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10 000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin,streptavidin, DNA, RNA, thyroxine binding globulin, thyroxine bindingprealbmin, transcortin, etc.

The term analyte further includes polynucleotide analytes such as thosepolynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA,DNA-ENA duplexes, etc. The term analyte also includes receptors that arepolynucleotide binding agents, such as, for example, restrictionenzymes, activators, repressors, nucleases, polymerases, histones,repair enzymes, chemotherapeutic agents, and the like.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

"Specific binding pair (sbp) member" refers to one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones-hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,and the like are not immunological pairs but are included in theinvention and the definition of sbp member.

"Polynucleotide" refers to a compound or composition which is apolymeric nucleotide having in the natural state about 6 to 500,000 ormore nucleotides and having in the isolated state about 6 to 50,000 ormore nucleotides, usually about 6 to 20,000 nucleotides, more frequently6 to 10,000 nucleotides. The term "polynucleotide" also includesoligonucleotides and nucleic acids from any source in purified orunpurified form, naturally occurring or synthetically produced,including DNA (dsDNA and ssDNA) and RNA, usually DNA, and may be t-RNA,m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA,DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, thegenomes of biological material such as microorganisms, e.g., bacteria,yeasts, viruses, viroids, molds, fungi, plants, animals, humans, andfragments thereof, and the like.

"Polynucleotide probe" refers to single-stranded nucleic acid moleculeshaving base sequences complementary to that of the target polynucleotideanalyte. Probes will generally consist of chemically or synthesized DNAor RNA polynucleotides from 6 to 200 base pair in length and must becapable of forming a stable hybridization complex with the targetpolynucleotide analyte.

"Ligand" refers to any organic compound for which a receptor naturallyexists or can be prepared. The term ligand also includes ligand analogs,which are modified ligands, usually an organic radical or analyteanalog, usually of a molecular weight greater than 100, which cancompete with the analogous ligand for a receptor, the modificationproviding means to join the ligand analog to another molecule. Theligand analog will usually differ from the ligand by more thanreplacement of a hydrogen with a bond which links the ligand analog to ahub or label, but need not. The ligand analog can bind to the receptorin a manner similar to the ligand. The analog could be, for example, anantibody directed against the idiotype of an antibody to the ligand.

"Receptor" or "antiligand" refers to any compound or composition capableof recognizing a particular spatial and polar organization of amolecule, e.g., epitopic or determinant site. Illustrative receptorsinclude naturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, avidin,protein A, barstar, complement component Clq, and the like. Avidin isintended to include egg white avidin and biotin binding proteins fromother sources, such as streptavidin.

"Specific binding" refers to the specific recognition of one of twodifferent molecules for the other compared to substantially lessrecognition of other molecules. Generally, the molecules have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the two molecules. Exemplary of specific binding areantibody-antigen interactions, enzyme-substrate interactions,polynucleotide interactions, and so forth.

"Non-specific binding" refers to the non-covalent binding betweenmolecules that is relatively independent of specific surface structures.Non-specific binding may result from several factors includinghydrophobic interactions between molecules.

"Antibody" refers to an immunoglobulin which specifically binds to andis thereby defined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera (polyclonal)or by preparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal), or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.Antibodies may include a complete immunoglobulin or fraent thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab')₂, Fab', and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

"Linking group" refers to the covalent linkage between molecules. Thelinking group will vary depending upon the nature of the molecules, suchas a photosensitizer, a photoactive indicator precursor, a sbp member orthe molecule associated with or part of a particle, being linked.Functional groups that are normally present or are introduced on aphotosensitizer or a photoactive indicator precursor will be employedfor linking these molecules to an sbp member or to a particle such as alipophilic component of a liposome or oil droplet, latex particle,silicon particle, metal sol, or dye crystallite.

For the most part, carbonyl functionalities are useful as linkinggroups, such as oxocarbonyl groups such as aldehydes, acetyl and carboxygroups; and non-oxocarbonyl groups (including nitrogen and sulfuranalogs) such as amidine, amidate, thiocarboxy and thionocarboxy.Alternative functionalities of oxo are also useful as linking groups,such as halogen, diazo, mercapto, olefin (particularly activatedolefin), amino, phosphoro and the like. A good description of linkinggroups may be found in U.S. Pat. No. 3,817,837, which disclosure isincorporated herein by reference.

The linking groups may vary from a bond to a chain of from 1 to 100atoms, usually from about 1 to 70 atoms, preferably 1 to 50 atoms, morepreferably 1 to 20 atoms, each independently selected from the groupnormally consisting of carbon, oxygen, sulfur, nitrogen and phosphorous.The number of heteroatoms in the linking groups will normally range fromabout 0 to 20, usually from about 1 to 15, more preferably 2 to 6. Theatoms in the chain may be substituted with atoms other than hydrogen ina manner similar to that described for organic groups. As a generalrule, the length of a particular linking group can be selectedarbitrarily to provide for convenience of synthesis, minimizeinterference of binding sbp members, and permit the incorporation of anydesired group such as a fluorescent energy acceptor, or a catalyst ofintersystem crossing such as a heavy atom, and the like. The linkinggroups may be aliphatic or aromatic, although with diazo groups,aromatic groups will usually be involved.

When heteroatoms are present, oxygen will normally be present as oxo oroxy, bonded to carbon, sulfur, nitrogen or phosphorous; nitrogen willnormally be present as nitro, nitroso or amino, normally bonded tocarbon, oxygen, sulfur or phosphorous; sulfur would be analogous tooxygen; while phosphorous will be bonded to carbon, sulfur, oxygen ornitrogen, usually as phosphonate and phosphate mono- or diester.

Common functionalities in forming a covalent bond between the linkinggroup and the molecule to be conjugated are alkylamine, amidine,thioamide, ether, urea, thiourea, guanidine, azo, thioether andcarboxylate, sulfonate, and phosphate esters, amides and thioesters.

For the most part, where the photosensitizer and the photoactiveindicator precursor of the present invention are linked to a particle,surface or sbp member, they will have a non-oxocarbonyl group (includingnitrogen and sulfur analogs), a phosphate group, an amino group, analkylating agent (e.g., such as halo or tosylalkyl), an ether group(including hydroxy), a thioether group (including mercapto), anoxocarbonyl group (e.g., aldehyde or ketone), or an active olefin suchas a vinyl sulfone or an α,β-unsaturated ester or amide. Thesefunctionalities will be linked to a particle, surface or a sbp memberhaving functionalities such as amine groups, carboxyl groups, activeolefins, or alkylating agents, e.g., bromoacetyl. Where an amine andcarboxylic acid or its nitrogen derivative or phosphoric acid arelinked, amides, amidines and phosphoramides will be formed. Wheremercaptan and activated olefin are linked, thioethers will be formed.Where a mercaptan and an alkylating agent are linked, thioethers will beformed. Where aldehyde and an amine are linked under reducingconditions, an alkylamine will be formed. Where a carboxylic acid orphosphate acid and an alcohol are linked, esters will be formed.

"A group or functionality imparting hydrophilicity or water solubility"refers to a hydrophilic functionality, which increases wettability ofsolids with water and the solubility in water of compounds to which itis bound. Such a functional group or functionality can be an organicgroup and can include a sulfonate, sulfate, phosphate, amidine,phosphonate, carboxylate, hydroxyl particularly polyols, amine, ether,amide, and the like. Illustrative functional groups are carboxyalkyl,sulfonoxyalkyl, CONHOCH₂ COOH, CO-(glucosamine), sugars, dextran,cyclodextrin, SO₂ NHCH₂ COOH, SO₃ H, CONHCH₂ CH₂ SO₃ H, PO₃ H₂, OPO₃ H₂,hydroxyl, carboxyl, ketone, and combinations thereof. Most of the abovefunctionalities can also be utilized as attaching groups, which permitattachment of the photosensitizer or photoactive indicator precursor toan sbp member or a support.

"A group or functionality imparting lipophilicity or lipid solubility"is a lipophilic functionality, which decreases the wettability ofsurfaces by water and the solubility in water of compounds to which itis bound. Such a functional group or functionality can contain 1 to 50or more atoms, usually carbon atoms substituted with hydrogen or halogenand can include alkyl, alkylidene, aryl and aralkyl. The lipophilicgroup or functionality will normally have one to six straight orbranched chain aliphatic groups of at least 6 carbon atoms, more usuallyat least 10 carbon atoms, and preferably at least 12 carbon atoms,usually not more than 30 carbon atoms. The aliphatic group may be bondedto rings of from 5 to 6 members, which may be alicyclic, heterocyclic,or aromatic.

"Photosensitizer" refers to a molecule which, for the purposes of thisinvention, can be excited to a metastable state, usually a tripletstate, which in the proximity of molecular oxygen can directly orindirectly transfer its energy to the oxygen with concomitant excitationof the oxygen to a highly reactive excited state of oxygen oftenreferred to as singlet oxygen or ¹ O₂ (¹ Δ_(g)). The photosensitizerwill usually be excited by the absorption of light or by an energytransfer from an excited state of a suitable donor but may also beexcited by chemiexcitation, electrochemical activation or by othermeans. Usually excitation of the photosensitizer will be caused byirradiation with light from an external source. The photosensitizers ofthis invention will usually have an absorption maximum in the wavelengthrange of 250-1100 nm, preferably 300-1000 nm, and more preferably450-950 nm, with an extinction coefficient at its absorbance maximumgreater than 500 M⁻¹ cm⁻¹, preferably at least 5000 M⁻¹ cm⁻¹, morepreferably at least 50,000 M⁻¹ cm⁻¹. The lifetime of the excited state,usually a triplet state, produced following absorption of light by thephotosensitizer will usually be at least 100 nsec, preferably at least 1μsec in the absence of oxygen. In general, the lifetime must besufficiently long to permit the energy transfer to oxygen, which willnormally be present at concentrations in the range of 10⁻⁵ to 10⁻³ M(depending on the medium). The excited state of the photosensitizer willusually have a different spin quantum number (S) than its ground stateand will usually be in a triplet (S=1) state when, as is usually thecase, the ground state is a singlet (S=0). Preferably, thephotosensitizer will have a high intersystem crossing yield. That is,excitation of a photosensitizer will produce the long lived state(usually triplet) with an efficiency of at least 10%, desirably at least40%, preferably greater than 80%. The photosensitizer will usually be atmost weakly fluorescent under the assay conditions (quantum yieldusually less than 0.5, preferably less that 0.1).

Photosensitizers of the instant invention are relatively photostable andwill not react efficiently with the singlet molecular oxygen sogenerated. Several structural features are present in most usefulphotosensitizers. Most photosensitizers have at least one and frequentlythree or more conjugated double or triple bonds held in a rigid,frequently aromatic structure. They will frequently contain at least onegroup that accelerates intereystem crossing such as a carbonyl or iminegroup or a heavy atom selected from rows 3-6 of the periodic table,especially iodine or bromine, or they will frequently have polyaromaticstructures. Typical photosensitizers include ketones such as acetone,benzophenone and 9-thioxanthone; xanthenes such as eosin and rosebengal; polyaromatic compounds such as buckminsterfullerene and9,10-dibromoanthracene; porphyrins including metallo-porphyrins such ashematoporphyrin and chlorophylis; oxazines; cyanines; squarate dyes;phthalocyanines; merocyanines; thiazines such as methylene blue, etc.,and derivatives of these compounds substituted by an organic group forenhancing intersystem crossing and rendering such compounds morelipophilic or more hydrophilic and/or as attaching groups forattachment, for example, to an sbp member. Examples of otherphotosensitizers that may be utilized in the present invention are thosethat have the above properties and are enumerated in N. J. Turro,"Molecular Photochemistry", page 132, W. A. Benjamin Inc., N.Y. 1965.

The photosensitizers of the instant invention are preferably relativelynon-polar to assure dissolution into a lipophilic member when thephotosensitizer is incorporated into a suspendible particle such as anoil droplet, liposome, latex particle, and the like.

"Photoactive indicator precursor" refers to those molecules which reactwith singlet oxygen to form photoactive indicators or to form a compoundthat can react with an auxiliary compound that is thereupon converted toa photoactive indicator. There are several types of reactions of singletotgen that can give compounds that will lead to a photoactive indicatorcompound. The type of reaction that is employed and the choice of thephotoactive indicator that is desired provides a guide to the structuresof the photoactive indicator precursors and any auxiliary compounds usedin the present invention.

The photoactive indicator precursor will preferably undergo a reactionwith singlet oxygen that is very rapid, usually at least 10⁴ -10⁶ sec⁻¹,preferably at least 10⁶ -10⁸ sec⁻¹, more preferably >10⁸ sec⁻¹. When theinitial product of the reaction is an intermediate that reacts to givethe photoactive precursor, the intermediate will preferably have alifetime that is short relative to the desired time between formingsinglet oxygen and detecting the fluorescence emitted from thephotoactive indicator upon exposure to light. For simultaneous singletoxygen generation and fluorescence detection the lifetime will usuallybe 10⁻³ -10 sec, preferably 10⁻³ sec. When generation of singlet oxygenand fluorescence detection are sequential the lifetime may vary from10⁻³ sec to 30 minutes or more, preferably <1 sec-60 sec.

Higher rates of reaction of singlet oxygen are achieved by providingsinglet oxygen reactive groups in the photoactive indicator precursorthat are electron rich. These groups will usually be an olefin oracetylene, hydrazine and hydroxylamine deroivatives, selenides andtellurides but are not limited to these groups. For example, tellurideshave been found to be particularly useful because they react rapidlywith singlet oxygen to produce an olefin. The reaction rate depends onthe electron availability (oxidation potential) of the tellurium. Forexample, electron donating groups on an aryl ring substituent on thetellurium atom can increase the rate. Changing from tellurium toselenium (the next lower chalcogenide) will decrease the rate, butincrease the oxidation stability of the molecule.

When the photoactive indicator precursor contains a hydrazine orhydrazide, reaction with singlet oxygen can produce a double bond. Forexample, singlet oxygen can convert hydrazides directly into fluorescentphotoactive indicators, as illustrated in the following reaction:##STR3##

The oxidation potential of a hydrazine is an important factor inproviding a high rate of reaction. Electron withdrawing groups such asan acyl group (e.g., as in a hydrazide) slow the reaction although acylhydrazides and diacyl hydrazides can still be used as photoactiveindicator precursors in the present invention. When the reaction isinsufficiently rapid it can often be accelerated in the presence of abase. For example, 3-aminophthaloyl hydrazide forms an anion in thepresence of strong base that is electron rich and can react rapidly withsinglet oxygen to form 3-aminophthalate as the photoactive indicator.However, the hydroxyl ion cannot be used as a base when the suspendibleparticles contain the photoactive indicator precursor within ahydrophobic matrix. Hydrophilic particles such as agarose can be usedinstead to permit access to the hydroxyl ion. Usually the photoactiveindicator precursor will be covalently bound to the suspendible particlewhen the particle is hydrophilic.

Still another example of a useful singlet oxygen reaction is thereaction with electron rich olefins such as those described in EuropeanPublished Patent Application No. 0 515 194. Two fundamental types ofreactions are described. One of these is the "ene" reaction which isexemplified by the following transformation: ##STR4## This reactionshifts the position of a double bond and produces a hydroperoxide. Thedouble bond shift can cause two auxochromic groups in the photoactiveindicator precursor to come into conjugation and thus produce afluorescent photoactive indicator.

Other photoactive indicator precursors react with singlet oxygen to formhydroperoxides which can react internally with an oxidizable group togive a fluorescent photoactive indicator. An example of such a precursorand the subsequent reaction and product include the following: ##STR5##

Alternatively, a hydroperoxide formed by reaction of singlet oxygen witha photoactive indicator precursor, such as 1,3-diphenylpropene, canserve to oxidize the leuco form of a dye which is present as anauxiliary compound so as to form a fluorescent photoactive indicator.The hydroperoxide can also oxygenate a group V element in an auxiliarycompound to cause it to act as an electron donating quencher of anassociated fluorescent group. For example, the auxiliary compound:##STR6## which is poorly fluorescent, can be oxygenated by ahydroperoxide to give the more highly fluorescent compound: ##STR7##

The auxiliary compound could alternatively have a selenium or telluriumatom that could react with a hydroperoxide to produce an intermediatethat could undergo subsequent elimination to form a fluorescentphotoactive indicator.

Alternatively, the photoactive indicator precursor will react slowly ornot at all with singlet oxygen but will react with a hydroperoxidereaction product of singlet oxygen and an auxiliary molecule. Forexample, in the following reaction, the auxiliary compound is reactedwith singlet oxygen to form a hydroperoxide, which is then reacted withthe photoactive indicator precursor of formula (Ik) to form afluorescent photoactive indicator: ##STR8##

In each of these examples the auxiliary compound and the photoactiveindicator precursor may be covalently linked. In such an occurrence, theresulting molecule is referred to as photoactive indicator precursor.

The second typical reaction of olefins with singlet oxygen is 2+2addition to form a dioxetane. This reaction can lead to bond breakingand therefore can separate a quenching group from a fundamentallyfluorescent molecule. Alternatively the bond breaking step can lead to aketone, aldehyde or ester which could be fluorescent or which couldundergo subsequent reactions that could lead to a fluorescent molecule.

In all of the above olefin reactions the rate will be faster if theolefin is substituted with electron donating groups such as ethers,thioethers, amines, and the like.

Still another type of reaction of singlet oxygen is 4+2 cycloadditionswith dienes. Such reactions lead initially to endoperoxides. In somecases endoperoxides can rearrange to active esters or anhydrides thatare capable of reaction with a suitably placed group to provide alactone or lactam that can be fluorescent. For example, the endoperoxideformed in the following reaction scheme can rearrange to form afluorescent lactone: ##STR9##

Alternatively, the endoperoxides may oxidize a photoactive indicatorprecursor much as described above for hydroperoxides.

Additional examples of photoactive indicator precursors' reaction withsinglet oxygen to produce fluorescent photoactive indicator moleculesare illustrated below: ##STR10##

The structure of the photoactive indicator precursor will thereforedepend on the particular singlet oxygen reaction that is to be employedand it will usually be designed to assure that any subsequent reactionsinitiated by reaction with singlet oxygen that are required to produce aphotoactive indicator will proceed relatively rapid. Additionally thestructure of the photoactive indicator precursor will lead to theformation of a photoactive indicator that has the desired absorption andemission wavelengths, and has relatively high fluorescent quantumyields, preferably >0.1, more preferably greater than 0.4, and a highextinction coefficient at the desired excitation wavelength,preferably >1000 M⁻¹ cm⁻¹, more preferably >10,000 M⁻¹ cm⁻¹.

Preferred photoactive indicator precursors of the present inventioninclude compounds containing the following structure (I): ##STR11##wherein H is cis to the XR group; X is a selenium or tellurium; R is anorganic or organometallic group bound to X through an unsaturated carbonatom, a silicon atom, or a tin atom; and A, when taken with thecarbon-carbon group, forms an alicyclic ring (optionally fused to one ormore aromatic rings) or a heterocyclic ring; where, upon reaction of thecompound with singlet oxygen, the H and the XR group are replaced by acarbon-carbon double bond to yield a fluorescent molecule having anextinction coefficient of at least 10,000 M⁻¹ cm⁻¹ at its absorptionmaximum and a fluorescence quantum efficiency of at least 10%.

Particularly preferred within these compounds are those compoundswherein X is tellurium. Most preferred is the compound of the followingformula: ##STR12## wherein R is an organic or organometallic group boundto X through an unsaturated carbon atom, a silicon atom, or a tin atom;and R¹ is hydrogen or alkyl; and wherein up to four of the remaininghydrogen atoms may be replaced by alkyl or alkylene substituents whichmay be taken together to form one or more alicyclic or aromatic rings.

The compounds disclosed herein containing structure (I) are designatedherein as derivatives of the structure, e.g., compound of formula (Ia),compound of formula (If) or compound of formula (Ik). Examples of suchcompounds where X is tellurium and the fluorescent photoactive indicatormolecule formed upon the compounds reaction with singlet oxygen aregiven below: ##STR13##

Presently, preferred photosensitive indicator precursors of theinvention are the compounds of formula (Ie) and (If) above.

The phenyltelluridyl radical (-TeC₆ H₅) in these compounds can bereplaced with other tellurium derivatives, such as TeSiC(CH₃)₃ andTeSn((CH₂)₃ CH₃)₃, or the phenyl group can be substituted, preferablywith electron donating groups such as -N(CH₃)₂ and -OCH₃. When X isselenium it is preferable that the selenium is substituted by a strongelectron donor group or atom, such as tin.

Other classes of photoactive indicator precursors can also be used inthe present invention. For example, compounds that chemiluminesce onreaction with singlet oxygen are frequently converted to fluorescentproducts which can serve as photoactive indicators of the presentinvention. Examples of such photoactive indicator precursors and thephotoactive indicators produced upon reaction with singlet oxygeninclude the following: ##STR14##

"Photoactive indicator" refers to a molecule which, following absorptionof light of wavelengths of 250 to 1100 nm, preferably 300 to 950 nm,emits light by fluorescence or phosphorescence, preferably byfluorescence, or transfers it excitation energy to an acceptor moleculewhich thereupon emits light by fluorescence or phosphorescence.Preferably the emission quantum yield will be high, usually at least0.1, preferably at least 0.4, more preferably greater than 0.7 and theextinction coefficient of the absorption maximum will usually be greaterthan 5000 M⁻¹ cm⁻¹.

Photoactive indicators of this invention are typically fluorescentcompounds, such as fluorescent brighteners, which typically absorb lightbetween 300 and 400 nanometers and emit between 400 and 500 nanometers;xanthenes such as rhodamine and fluorescein; bimanes; coumarins such asumbelliferone; aromatic amines such as dansyl; squarate dyes;benzofurans; cyanines, merocyanines, rare earth chelates, and the like.Photoactive indicators that are phosphorescent include porphyrins,phthalocyanines, polyaromatic compounds such as pyrene, anthracene andacenaphthene. Photoactive indicators also include chromenes. Photoactiveindicators that can transfer energy to an acceptor molecule will usuallyabsorb at 250 to 550 nm. Such acceptor molecules are luminescent and caninclude any of the above mentioned fluorescent and phosphorescentphotoactive indicators.

"Measuring the fluorescence" refers to the detection and calculation ofthe amount of light emitted from a photoactive indicator of theinvention upon excitation by irradiation with light. While thefluorescence of the photoactive indicator will usually be measured byexciting the photoactive indicator by irradiation with light andsimultaneously detecting the light that is emitted therefrom (i.e., thefluorescence), other methods of detecting the fluorescence arecontemplated by this invention. The measurement of fluorescence isintended to include detection of light emitted by the photoactiveindicator simultaneous with or immediately following irradiation withlight regardless of whether the light is absorbed directly or indirectlyor whether the emission is from an excited singlet state or state ofhigher multiplicity. Measurement of fluorescence is also intended toinclude the measurement of light emitted from the photoactive indicatorfollowing transfer of energy from a donor that is excited throughchemiexcitation other than chemiexcitation initiated by absorption oflight by the photosensitizer. For example, measurement of fluorescenceof the photoactive indicator includes activation of a chemiluminescentmolecule, for example, by addition of hydrogen peroxide and peroxidaseto luminol, and measurement of the light emitted from the photoactiveindicator as a result of the energy transfer from the luminol reactionproduct to the photoactive indicator.

"Support" or "surface" refers to a surface comprised of porous ornon-porous water insoluble material. The surface can have any one of anumber of shapes, such as strip, rod, particle, including bead, and thelike. The surface can be hydrophilic or capable of being renderedhydrophilic and includes inorganic powders such as silica, magnesiumsulfate, and alumina; natural polymeric materials, particularlycellulosic materials and materials derived from cellulose, such as fibercontaining papers, e.g., filter paper, chromatographic paper, etc.;synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, lipid vesicles, andcells can also be employed.

Binding of sbp members to the support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).

The surface will usually be polyfunctional or be capable of beingpolyfunctionalized or be capable of binding a polynucleotide, an sbpmember, a photosensitizer, and/or a photoactive chemiluminescentcompound through specific or non-specific covalent or non-covalentinteractions. A wide variety of functional groups are available or canbe incorporated. Functional groups include carboxylic acids, aldehydes,amino groups, cyano groups, ethylene groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto surfaces is well know and is amply illustrated in the literature. Seefor example Cautrecasas, J. Biol. Chem. 245,3059 (1970). The length of alinking group to the oligonucleotide or sbp member may vary widely,depending upon the nature of the compound being linked, the effect ofthe distance between the compound being linked and the surface on thespecific binding properties and the like.

"Suspendible particles" refers to particles capable of being able to besuspended in water which are at least about 20 nm and not more thanabout 20 microns, usually at least about 40 nm and less than about 10microns, preferably from about 0.10 to 2.0 microns in diameter, andwhich normally have a volume of less than about 4 picoliters. Thesuspendible particles may be organic or inorganic, swellable ornon-swellable, porous or non-porous, preferably of a densityapproximating water (generally from about 0.7 to about 1.5 g/ml), andcomposed of material that can be transparent, partially transparent, oropaque. The suspendible particles will usually be charged, preferablynegatively charged. The suspendible particles may be solid (e.g.,polymer, metal, glass, organic and inorganic such as minerals, salts anddiatoms), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon fluid),or vesicles (e.g., synthetic such as phospholipid or other lipids suchas dialkyl phosphates or natural such as cells and organelles). Thesuspendible particles may be latex particles or other particlescomprised of organic or inorganic polymers; lipid vesicles, e.g.,liposomes; phospholipid vesicles; oil droplets; silicon particles; metalsols; cells; and dye crystallites.

If organic, the suspendible particles may be polymers, either additionor condensation polymers, which are readily suspendible in the assaymedium. The organic suspendible particles will also be adsorptive orfunctionalizable so as to bind at their surface an sbp member (eitherdirectly or indirectly) and to bind at their surface or incorporatewithin their volume a photosensitizer or a photoactive indicatorprecursor.

The suspendible particles can be derived from naturally occurringmaterials, naturally occurring materials which are syntheticallymodified and synthetic materials. Natural or synthetic assemblies suchas lipid bilayers, e.g., liposomes and non-phospholipid vesicles, arepreferred. Among organic polymers of particular interest arepolysaccharides, particularly cross-linked polysaccharides, such asagarose, which is available as Sepharose, dextran, available as Sephadexand Sephacryl, cellulose, starch, and the like; addition polymers, suchas polystyrene, polyacrylamide, homopolymers and copolymers ofderivatives of acrylate and methacrylate, particularly esters and amideshaving free hydroxyl functionalities including hydrogels, and the like.Inorganic polymers include silicones, glasses, available as Bioglas, andthe like. Sols include gold, selenium, and other metals. Suspendibleparticles may also be dispersed water insoluble dyes such as porphyrins,phthalocyanines, etc., which may also act as photosensitizers.Suspendible particles may also include diatoms, cells, viral particles,oil droplets, fat particles such as alkyl triglycerides, magnetosomes,cell nuclei and the like.

Where non-polymeric particles are used, the particle size may be variedby breaking larger particles into smaller particles by mechanical means,such as grinding, sonication, agitation, etc.

Like the surface or support defined above, the suspendible particleswill usually be polyfunctional or be capable of being polyfunctionalizedor be capable of being bound to an sbp member, photosensitizer, orphotoactive indicator precursor through specific or non-specificcovalent or non-covalent interactions. A wide variety of functionalgroups are available or can be incorporated. Exemplary functional groupsinclude carboxylic acids, aldehydes, amino groups, cyano groups,ethylene groups, hydroxyl groups, mercapto groups and the like. Whencovalent attachment of a sbp member, chemiluminescent compound orphotosensitizer to the particle is employed, the manner of linking iswell known and is amply illustrated in the literature. See for exampleCautrecasas, J. Biol. Chem., 245:3059 (1970). The length of a linkinggroup may vary widely, depending upon the nature of the compound beinglinked, the nature of the particle, the effect of the distance betweenthe compound being linked and the particle on the binding of sbp membersand the analyte and the like.

The photosensitizer and/or photoactive indicator precursor can be chosento dissolve in, or covalently or bind to suspendible particles,provided, however, that the photosensitizer and the photoactiveindicator precursor are not associated with the same particle. Whennoncovalently bound, the compounds and the particles will all usually behydrophobic to reduce the ability of the compounds to dissociate fromthe particles and to associate with the same particle. The problem ofhaving both the photosensitizer and the photoactive indicator associatedwith the same particle may be minimized by covalently binding either oneor both of the compounds to a particle, thereby allowing each compoundto be either hydrophilic or hydrophobic.

The number of photosensitizer or photoactive indicator precursormolecules associated with each particle will be at least one and may besufficiently high enough so that the particle consists entirely ofphotosensitizer or photoactive indicator precursor molecules. Thepreferred number of molecules will be selected empirically to providethe highest signal to background in the assay (where the signal isdetermined under conditions where the particles are bound to each otherand the background is determined where the particles are unassociated).Normally, the concentration of photosensitizer and photoactive indicatorprecursor in the particles will range from 10⁻⁸ to 5M, usually from 10⁻⁵to 10⁻¹ M, preferably from 10⁻³ to 10⁻¹ M.

"Oil droplets" refers to fluid or waxy particles comprised of alipophilic compound coated and stabilized with an emulsifier that is anamphiphilic molecule such as, for example, phospholipids, sphingomyelin,albumin and the like.

The phospholipids are based upon aliphatic carboxylic acid esters ofaliphatic polyols, where at least one hydroxylic group is substitutedwith a carboxylic acid ester of from about 8 to 36, more usually of fromabout 10 to 20 carbon atom, which may have from 0 to 3, more usuallyfrom 0 to 1 site of ethylenic unsaturation and at least 1, normally only1, hydroxyl group substituted with phosphate to form a phosphate ester.The phosphate group may be further substituted with small aliphaticcompounds which are of di or higher functionality, generally havinghydroxyl or amino groups.

The oil droplets can be made in accordance with conventional proceduresby combining the appropriate lipophilic compounds with a surfactant,anionic, cationic or nonionic, where the surfactant is present in fromabout 0.1 to 40, more usually from about 0.1 to 20 weight percent of themixture and subjecting the mixture in an aqueous medium to agitation,such as sonication or vortexing. Illustrative lipophilic compoundsinclude hydrocarbon oils, halocarbons including fluorocarbons, alkylphthalates, trialkyl phosphates, triglycerides, etc.

An sbp member will usually be adsorbed to the surface of the oil dropletor bonded directly or indirectly to a surface component of the oildroplet. The sbp member may be incorporated into the liquid particleseither during or after the preparation of the liquid particles. The sbpmember will normally be present in from about 0.5 to 100, more usually 1to 90, frequently from about 5 to 80 and preferably from about 50 to 100mole percent of the molecules present on the surface of the particle.

The following is a list, by way of illustration and not limitation, ofamphiphilic compounds, which may be utilized for stabilizing oildroplets: phosphatidyl ethanolamine, phosphatidyl choline, phosphatidylserine, dimyristoylphosphatidyl choline, egg phosphatidyl choline,diapalmitoylphosphatidyl choline, phosphatidic acid, cardiolipin,lecithin, galactocerebroside, sphingomyelin, dicetylphosphate,phosphatidyl inositol, 2-trihexadecylammoniumethylamine,1,3-bis(octadecyl phosphate)-propanol, stearoyloxyethylene phosphate,phospholipids, dialkylphosphates, sodium dodecyl sulfate, cationicdetergents, anionic detergents, proteins such as albumin, non-ionicdetergents, etc.

Stabilization of oil droplets can also be achieved by coating with apolymer such as polycyanoacrylates, dextran, polymerized proteins suchas albumin, hydroxybutyl methacrylate, polyacrylamide and the like.

Other compounds may also be used which have lipophilic groups and whichhave been described previously. For the most part, these compounds willbe alkylbenzenes, having alkyl groups of from 6 to 20 carbon atoms,usually mixtures of alkyl groups, which may be straight or branchedchain, and having a carboxyl group, an hydroxylic group, a polyoxyalkylene group (alkylene of from 2 to 3 carbon atoms), carboxylic group,sulfonic acid group, or amino group. Aliphatic fatty acids may be usedwhich will normally be of from about 10 to 36, more usually of fromabout 12 to 20 carbon atoms. Also, fatty alcohols having the carbonlimits indicated for the fatty acids, fatty amines of similar carbonlimitations and various steroids may also find use.

The oil droplets can comprise a fluorocarbon oil or a silicone oil(silicon particle). Such droplets are described by Giaever in U.S. Pat.Nos. 4,634,681 and 4,619,904 (the disclosures of which are incorporatedherein in their entirety). These droplets are formed by dispersing afluorocarbon oil or silicone oil in an aqueous phase. The droplets areprepared by placing a small amount of the selected oil (generally, suchoils are commercially available) in a container with a larger amount ofthe aqueous phase. The liquid system is subjected to agitation to bringabout emulsification and then centrifuged. The homogeneous phase isremoved and the residual droplets are resuspended in an aqueous bufferedmedium. The above centrifugation and decantation steps can be repeatedone or more times before the droplets are utilized.

Sbp members can be bound to the droplets in a number of ways. Asdescribed by Giaever, the particular sbp member, particularly aproteinaceous sbp member, can be coated on the droplets by introducingan excess of the sbp member into the aqueous medium prior to or afterthe emulsification step. Washing steps are desirable to remove excesssbp member. Functionalization of the oil introduces functionalitiesdescribed above for linking to sbp members. Such functionalities canalso be employed to link the droplets to a photosensitizer or aphotoactive indicator precursor. On the other hand, the photosensitizeror photoactive indicator precursor will frequently be chosen to besoluble in the oil phase of the oil droplet and will not be covalentlybound. When the oil is a fluorocarbon, a fluorinated photosensitizer orphotoactive indicator precursor will often be more soluble than thecorresponding unfluorinated derivation.

"Liposomes" refers to microvesicles of approximately spherical shape andare one of the preferred materials for use in the present invention. Theliposomes have a diameter that is at least about 20 nm and not more thanabout 20 microns, usually at least about 40 nm and less than about 10microns. Preferably, the diameter of the liposomes will be less thanabout two microns so as to limit settling or floatation.

The outer shell of a liposome consists of an amphiphilic bilayer thatencloses a volume of water or an aqueous solution niposomes with morethan one bilayer are referred to as multilamellar vesicles. Liposomeswith only one bilayer are called unilamellar vesicles. Multilamellarvesicles are preferred in the present invention when using a lipophilicphotosensitizer or photoactive indicator precursor because of theirability to incorporate larger quantities of these materials thanunilamellar vesicles. The amphiphilic bilayer is frequently comprised ofphospholipids. Phospholipids employed in preparing particles utilizablein the present invention can be any phospholipid or phospholipid mixturefound in natural membranes including lecithin, or synthetic glycerylphosphate diesters of saturated or unsaturated 12-carbon or 24-carbonlinear fatty acids wherein the phosphate can be present as a monoester,or as an ester of a polar alcohol such as ethanolamine, choline,inositol, serine, glycerol and the like. Particularly preferredphospholipids include L-α-palmitoyl oleoyl-phosphatidylcholine (POPC),palmitoyl oleoylphosphatidyl-glycerol (POPG),L-α-dioleoylphosphatidylglycerol, L-α-(dioleoyl)-phosphatidylethanolamine (DOPE) and L-α-(dioleoyl)-phosphatidylβ-(4-(N-maleimidomethyl)-cyclohexane-1-carboxyamido)ethanol (DOPE-MCC).

The phospholipids in the bilayer may be supplemented with cholesteroland may be replaced with other amphiphilic compounds that have a polarhead group, usually charged, and hydrophobic portion usually comprisedof two linear hydrocarbon chains. Examples of such substituents includedialkylphosphate, dialkoxypropylphosphates wherein the alkyl groups havelinear chains of 12-20 carbon atoms,N-(2,3-di(9-(Z)-octa-decenyloxy))-prop-1-yl-N,N,N-trimethyl-ammoniumchloride (DOTMA), sphingomyelin, cardiolipin, and the like.

Liposomes utilized in the present invention preferably have a highnegative charge density to stabilize the suspension and to preventspontaneous aggregation.

Liposomes may be produced by a variety of methods including hydrationand mechanical dispersion of dried phospholipid or phospholipidsubstitute in an aqueous solution. Liposomes prepared in this mannerhave a variety of dimensions, compositions and behaviors. One method ofreducing the heterogeneity and inconsistency of behavior of mechanicallydispersed liposomes is by sonication. Such a method decreases theaverage liposome size. Alternatively, extrusion is usable as a finalstep during the production of the liposomes. U.S. Pat. No. 4,529,561discloses a method of extruding liposomes under pressure through auniform pore-size membrane to improve size uniformity.

For use in the present invention the liposomes should be capable ofbinding to an sbp member and be capable of having a photosensitizer orphotoactive indicator precursor associated with either the aqueous orthe nonaqueous phase. The liposomes utilized in the present inventionwill usually have sbp members bound to the outer surface of the lipidvesicle.

Preparation of liposomes containing a hydrophobic or amphiphilicphotosensitizer or a photoactive indicator precursor dissolved in thelipid bilayer can be carried out in a variety of methods, including amethod described by Olsen, et al., Biochemica et Biophysica Acta,557(9), 1979. Briefly, a mixture of lipids containing the appropriatecompound in an organic solvent such as chloroform is dried to a thinfilm on the walls of a glass vessel. The lipid film is hydrated in anappropriate buffer by shaking or vortexing. Thereafter, the lipidsuspension is extruded through a series of polycarbonate filtermembranes having successively smaller pore sizes, for example, 2.0, 1.0,0.8, 0.6, 0.4, and 0.2 microns. Repeated filtration through any of thefilters, and in particular through the smallest filter, is desirable.The liposomes can be purified by, for example, gel filtration, such asthrough a column of Sephacryl S-1000. The column can be eluted withbuffer and the liposomes collected. Storage in the cold prolongsshelf-life of the liposomes produced by this method. Alternatively thephotosensitizer or photoactive indicator precursor can be added to theliquid suspension following preparation of the liposomes.

Labeling of droplets and liposomes will often involve, for example,inclusion of thiol or maleimide or bionin groups on the moleculescomprising the lipid bilayer. Photosensitizers, photoactive indicatorprecursor molecules or sbp members may then be bound to the surface byreaction of the particles with one of these materials that is bound to asulfhydryl reactive reagent, a sulfhydryl group, or avidin,respectively. Sulfhydryl reactive groups include alkylating reagentssuch as bromoacetamide and maleimide.

Sbp members can be attracted to the surface of the liposome particles byweak hydrophobic interactions, however such interactions are notgenerally sufficient to withstand the shear force encountered duringincubation and washing. It is preferable to covalently bond sbp membersto a liposome particle that has been functionalized, for example by useof DOPE-MCC, as shown above, by combining said liposome with theselected sbp member functionalized with a mercaptan group. For example,if the sbp member is an antibody, it may be reacted withS-acetyl-mercaptosuccinic anhydride (SAMSA) and hydrolyzed to provide asulfhydryl modified antibody.

"Latex particles" refers to a particulate water-suspendiblewater-insoluble polymeric material usually having particle dimensions of20 nm to 20 mm, more preferably 100 to 1000 run in diameter. The latexis frequently a substituted polyethylene such as the following:polystyrene-butadiene, polyacrylamide polystyrene, polystyrene withamino groups, poly-acrylic acid, polymethacrylic acid,acrylonitrile-butadiene, styrene copolymers, polyvinyl acetate-acrylate,polyvinyl pyridine, vinyl-chloride acrylate copolymers, and the like.Non-crosslinked polymers of styrene and carboxylated styrene or styrenefunctionalized with other active groups such as amino, hydroxyl, haloand the like are preferred. Frequently, copolymers of substitutedstyrenes with dienes such as butadiene will be used.

The association of the photosensitizer or photoactive indicatorprecursor with latex particles utilized in the present invention mayinvolve incorporation during formation of the particles bypolymerization but will usually involve incorporation into preformedparticles, usually by noncovalent dissolution into the particles.Usually a solution of the photoactive indicator precursor orphotosensitizer will be employed. Solvents that may be utilized includealcohols (including ethanol), ethylene glycol and benzyl alcohol; amidessuch as dimethyl formamide, formamide, acetamide and tetramethyl ureaand the like; sulfoxides such as dimethyl sulfoxide and sulfolane; andethers such as carbitol, ethyl carbitol, dimethoxy ethane and the like,and water. The use of solvents having high boiling points in which theparticles are insoluble permits the use of elevated temperatures tofacilitate dissolution of the compounds into the particles and areparticularly suitable. The solvents may be used singly or incombination. Particularly preferred solvents for incorporating aphotosensitizer are those that will not quench the triplet excited stateof the photosensitizer either because of their intrinsic properties orbecause they can subsequently be removed from the particles by virtue oftheir ability to be dissolved in a solvent such as water that isinsoluble in the particles. Aromatic solvents are preferred, andgenerally solvents that are soluble in the particle. For incorporatingphotoactive indicator precursors in particles a solvent should beselected that does not interfere with the fluorescence of thephotoactive indicator so formed because of their intrinsic properties orability to be removed from the particles. Frequently, aromatic solventswill also be preferred. Typical aromatic solvents includedibutylphthalate, benzonitrile, naphthonitrile, dioctylterephthalate,dichlorobenzene, diphenylether, dimethoxybenzene, etc.

Except when the photosensitizer or photoactive indicator precursor is tobe covalently bound to the particles, it will usually be preferable touse electronically neutral photosensitizers or photoactive indicatorprecursors. It is preferable that the liquid medium selected does notsoften the polymer beads to the point of stickiness. A preferredtechnique comprises suspending the selected latex particles in a liquidmedium in which the photosensitizer or photoactive indicator precursorhas at least limited solubility. Preferably, the concentrations of thephotosensitizer and photoactive indicator precursor in the liquid mediawill be selected to provide particles that have the highest efficiencyof singlet oxygen formation and highest quantum yield of emission fromthe photoactive indicator so formed in the media but less concentratedsolutions will sometimes be preferred. Distortion or dissolution of theparticles in the solvent can be prevented by adding a miscible cosolventin which the particles are insoluble.

Generally, the temperature employed during the procedure will be chosento maximize the singlet oxygen formation ability of thephotosensitizer-labeled particles and the quantum yield of thephotoactive indicator so formed from the photoactive indicatorprecursor-labelled particles with the proviso that the particles shouldnot become aggregated at the selected temperature. Elevated temperaturesare normally employed. The temperatures for the procedure will generallyrange from 20° C. to 200° C., more usually from 50° C. to 170° C. It hasbeen observed that some compounds that are nearly insoluble in water atroom temperature, are soluble in, for example, low molecular weightalcohols, such as ethanol and ethylene glycol and the like, at elevatedtemperatures. Carboxylated modified latex particles have been shown totolerate low molecular weight alcohols at such temperatures.

An sbp member may be physically adsorbed on the surface of the latexparticle or may be covalently bonded to the particle. In cases whereinthe sbp member is only weakly bound to the surface of the latexparticle, the binding may in certain cases be unable to endureparticle-to-particle shear forces encountered during incubation andwashings. Therefore, it is usually preferable to covalently bond sbpmembers to the latex particles under conditions that will minimizeadsorption. This may be accomplished by chemically activating thesurface of the latex. For example, the N-hydroxysuccinimide ester ofsurface carboxyl groups can be formed and the activated particles toreduce nonspecific binding of assay components to the particle surfaceare then contacted with a linker having amino groups that will reactwith the ester groups or directly with an sbp member that has an aminogroup. The linker will usually be selected to reduce nonspecific bindingof assay components to the particle surface and will preferably providesuitable functionality for both attachment to the latex particle andattachment of the sbp member. Suitable materials include maleimidatedaminodextran (MAD), polylysine, aminosaccharides, and the like. MAD canbe prepared as described by Hubert, et al., Proc. Natl. Acad. Sci.,75(7), 3143, 1978.

In one method, MAD is first attached to carboxyl-containing latexparticles using a water soluble carbodiimide, for example,1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. The coated particles arethen equilibrated in reagents to prevent nonspecific binding. Suchreagents include proteins such as bovine gamma globulin (BGG), anddetergent, such as Tween 20, TRITON X-100 and the like. A sbp memberhaving a sulfhydryl group, or suitably modified to introduce asulfhydryl group, is then added to a suspension of the particles,whereupon a covalent bond is formed between the sbp member and the MADon the particles. Any excess unreacted sbp member can then be removed bywashing.

"Metal sols" refers to those suspendible particles comprised of a heavymetal, i.e., a metal of atomic number greater than 20 such as a Group IBmetal, e.g., gold or silver.

Metal sol particles are described, for example, by Leuvering in U.S.Pat. No. 4,313,734, the disclosure of which is incorporated herein byreference in its entirety. Such sols include colloidal aqueousdispersion of a metal, metal compound, or polymer nuclei coated with ametal or metal compound.

The metal sols may be of metals or metal compounds, such as metaloxides, metal hydroxides and metal salts or of polymer nuclei coatedwith metals or metal compounds. Examples of such metals are platinum,gold, silver mercury, lead, palladium, and copper, and of such metalcompounds are silver iodide, silver bromide, copper hydrous oxide, ironoxide, iron hydroxide or hydrous oxide, aluminum hydroxide or hydrousoxide, chromium hydroxide or hydrous oxide, vanadium oxide, arsenicsulphide, manganese hydroxide, lead sulphide, mercury sulphide, bariumsulphate and titanium dioxide. In general, the metals or metal compoundsuseful may be readily demonstrated by means of known techniques.

It is sometimes advantageous to use sols comprised of dispersedparticles consisting of polymer nuclei coated with the above mentionedmetals or metal compounds. These particles have similar properties asthe dispersed phase of pure metals or metal compounds, but size, densityand metal contact can be optimally combined.

The metal sol particles may be prepared in a large number of ways whichare in themselves known. For example, for the preparation of a gold solLeuvering refers to an article by G. Frens in Nature Physical Science241, 20 (1973).

The metal sol particles can be modified to contain various functionalgroups as described above for linking to an sbp member or aphotosensitizer or a photoactive indicator precursor. For example,polymeric bonding agents can be used to coat the particles such aspolymers containing thiol groups that bond strongly to many heavy metalsor silylating agents that can bond and form polymeric coatings as, forexample, by reaction of metal particles with trialkoxy aminoalkylsilanesas described in European Published Patent Application 84400952.2 byAdvanced Magnetics for coating magnetic particles.

"Dye crystallites" refers to microcrystals of pure or mixed solid waterinsoluble dyes, preferably dyes that can serve as the photosensitizersdescribed above. The dye crystallites useful in the present inventionhave a size range of 20 nm to 20 μm.

One method for preparing dye crystallites is described in U.S. Pat. No.4,373,932 (Gribnau, et al.), the disclosure of which is incorporatedherein by reference in its entirety. Gribnau describes colloidal dyeparticles and aqueous dispersions of a hydrophobic dye or pigment, whichmay have an immunochemically reactive component directly or indirectlyattached. The dye particles are prepared in general by dispersing a dyein water and then centrifuging. A dye pellet is obtained and resuspendedin water, to which glass beads are added. This suspension is rolled forseveral days at room temperature. The liquid is decanted andcentrifuged, and the dye particles are obtained after aspiration of theliquid.

Another method for preparing dye crystallites is by slow addition of asolution of the dye in a water miscible solvent to water. Another methodis by sonication of a suspension of the solid dye in water.

Binding of sbp members to the dye particles can be achieved by direct orindirect adsorption or covalent chemical attachment. The latter isgoverned by the presence of suitable functional groups in any coatingmaterial and in the dye. For example, functional groups can. beintroduced onto the surface of a dye crystalline by coupling a compoundcontaining a diazotized aromatic amino group and the desired functionalgroup to a phenolic or anilino group of the dye.

Where the dye has a carboxyl group, the dye crystallite can be activatedby a carbodiimide and coupled to a primary amino component. Aliphaticprimary amino groups and hydroxyl groups can be activated, for example,by cyanogen bromide or halogen-substituted di- or tri-azines, afterwhich attachment with a primary amino component or, for example, with acomponent containing a --SH or --OH group can take place. Use can alsobe made of bifunctional reactive compounds. For example, glutaraldehydecan be used for the mutual coupling of primary amino components of thedye and an sbp member, and, for example, a hetero-bifunctional reagentsuch as N-succinimidyl 3-(2-pyridyldithio) propionate can be employedfor the coupling of a primary amino component to a component containinga thiol group.

"Wholly or partially sequentially" refers to the condition when thecomponents of the methods of the present invention are combined otherthan concomitantly (simultaneously), one or more may be combined withone or more of the remaining agents to form a subcombination. Eachsubcombination can then be subjected to one or more steps of the presentmethod. Thus, each of the subcombinations can be incubated underconditions to achieve one or more of the desired results.

Various ancillary materials will frequently be employed in the assay inaccordance with the present invention. For example, buffers willnormally be present in the assay medium, as well as stabilizers for theassay medium and the assay components. Frequently, in addition to theseadditives, proteins may be included, such as albumins, organic solventssuch as formamide, quaternary ammonium salts, polycations such asdextran sulfate, surfactants, particularly non-ionic surfactants,binding enhancers, e.g., polyalkylene glycols, or the like.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In general, the present invention is directed to methods for determiningan analyte in a selected medium. The methods comprise treating a mediumsuspected of containing an analyte under conditions such that theanalyte, if present, affects the amount of a photosensitizer and aphotoactive indicator precursor that can come into close proximitywherein the short-lived singlet oxygen generated by the photosensitizercan react with the photoactive indicator precursor prior to thespontaneous decay of the singlet oxygen in order to form a photoactiveindicator. The method further comprises exposing the photoactiveindicator to light which may be of the same or a different wavelengththat the light used to excite the photosensitizer in order to excite thephotoactive indicator so formed and then measuring the intensity offluorescence emitted from the photoactive indicator upon excitation. Theintensity of fluorescence produced is related to the amount of analytein the medium. The photoactive indicator is formed upon reaction of thephotoactive indicator precursor with the singlet oxygen generated by thephotosensitizer. The photosensitizer catalyzes the generation of singletoxygen usually in response to photoexcitation followed by energytransfer to molecular oxygen. Often one or both of the photosensitizerand the photoactive indicator precursor will be associated withsurfaces, wherein, in homogeneous assays, the surface will preferably bethe surface of suspendible particles.

For homogeneous assays the invention is predicated on an analyte causingor inhibiting molecules of the photosensitizer and the photoactiveindicator precursor to be closer to each other than their averagedistance in the bulk solution of the assay medium. The amount of thispartitioning will depend upon the amount of analyte present in thesample to be analyzed. The photosensitizer molecules that do not becomeassociated with the photoactive indicator precursor produce singletoxygen that is unable to reach the photoactive indicator precursorbefore undergoing decay in the aqueous medium. However, when thephotosensitizer and the photoactive indicator precursor come in closeproximity with each other in response to the presence of the analyte,the singlet oxygen produced upon irradiation of the photosensitizer canreact with the photoactive indicator precursor to form a photoactiveindicator before undergoing decay. Because numerous photoactiveindicator precursor molecules and/or photosensitizer molecules can beassociated with a surface or can be incorporated into the materialcomprising the surface, the presence of a surface in conjunction withthe photosensitizer and photoactive indicator precursor can increase theefficiency of, or action of, singlet oxygen with the photoactiveindicator precursor prior to decay. It is therefore preferred to bringone member of the photoactive indicator precursor and photosensitizerpair into the proximity of a surface that incorporates the other memberas a function of the presence of an analyte. The subject assay providesfor a convenient method for detecting and measuring a wide variety ofanalytes in a simple, efficient, reproducible manner, which can employsimple equipment for measuring the amount of light produced during thereaction.

The amount of photosensitizer that comes in close proximity to thephotoactive indicator precursor is affected by the presence of analyteby virtue of the photosensitizer and photoactive indicator precursoreach being associated with an sbp member. This may be accomplished in anumber of ways and the term "associated with" is defined thereby. Thephotosensitizer and photoactive indicator precursor may containfunctionalities for covalent attachment to sbp members and the sbpmembers may contain functionalities for attaching to the photosensitizerand/or photoactive indicator precursor. The attachment may beaccomplished by a direct bond between the two molecules or through alinking group which can be employed between an sbp member and thephotosensitizer or photoactive indicator precursor. In anotherembodiment either or both of the photosensitizer and photoactiveindicator precursor can be bound to surfaces or incorporated inparticles, to which are also attached sbp members. In both cases each ofthe sbp members is capable of binding directly or indirectly to theanalyte or an assay component whose concentration is affected by thepresence of the analyte. Either or both of the photosensitizer andphotoactive indicator precursor can be incorporated into particles byvirtue of being dissolved in at least one phase of the particles, inwhich case the solute will be at much higher concentration within theparticle than in the bulk assay medium.

Alternatively, either or both of the photosensitizer and photoactiveindicator precursor my be covalently bound to particles, either byproviding linking functional groups on the components to be bound or byincorporating the photosensitizer or photoactive indicator precursorinto a polymer that comprises the particles. For particles that are oildroplets or liposomes the photosensitizer and photoactive indicatorprecursor can be attached to one or more long hydrocarbon chains, eachgenerally having at least 10 to 30 carbon atoms. If the particles aredroplets of a fluorocarbon, the photosensitizer or photoactive indicatorprecursor incorporated into these particles may be fluorinated toenhance solubility and reduce exchange into other particles bound withthe other label, and the hydrocarbon chain used for linking willpreferably be replaced with a fluorocarbon chain. For silicon fluidparticles the photosensitizer and photoactive indicator precursor can bebound to a polysiloxane. In order to maximize the number ofphotosensitizer or photoactive indicator precursor molecules perparticle, it will usually be desirable to minimize the charge andpolarity of the photosensitizer or photoactive indicator precursor sothat it resides within the non-aqueous portion of the particle. When theparticle is a liposome and it is desired to retain the photosensitizeror photoactive indicator precursor in the aqueous phase of the liposome,it will be preferred to use photosensitizers or photoactive indicatorprecursors that are highly polar or charged.

No matter how the photosensitizer and the photoactive indicatorprecursor are associated with their respective sbp member, it iscritical that neither compound is capable of dissociating from its sbpmember and becoming associated with the sbp member associated with theother member of the photosensitizer and photoactive indicator precursorpair during the course of the assay. Thus, dissociation of thesecompounds from their respective sbp members must be slow relative to thetime required for the assay.

The photoactive indicator precursor may be bound to a sbp member that iscapable of binding directly or indirectly to the analyte or to an assaycomponent whose concentration is affected by the presence of theanalyte. The term "capable of binding directly or indirectly" means thatthe designated entity can bind specifically to the entity (directly) orcan bind specifically to a specific binding pair member or to a complexof two or more sbp members which is capable of binding the other entity(indirectly).

The surface generally has an sbp member bound to it. Preferably, thephotoactive indicator precursor is associated with the surface, usuallywithin a suspendible particle. This sbp member is generally capable ofbinding directly or indirectly to the analyte or a receptor for theanalyte. When the sbp members associated with the photosensitizer andthe photoactive indicator precursor are both capable of binding to theanalyte, a sandwich assay protocol can be used. When one of the sbpmembers associated with the photosensitizer or photoactive indicatorprecursor can bind both the analyte and an analyte analog, a competitiveassay protocol can be used. The attachment to a surface or incorporationin a particle of the photoactive indicator precursor is governedgenerally by the same principles described above for the attachment to,or the incorporation into, a particle of the photosensitizer.

The photosensitizer is usually caused to activate the photoactiveindicator precursor by irradiating the medium containing thesereactants. Since it will frequently be undesirable to excite thephotoactive indicator precursor directly with light, the wavelength oflight used to activate the photosensitizer will usually be longer thanthe longest wavelengths absorbed substantially by the photoactiveindicator precursor. However, the medium must be irradiated with a shortenough wavelength of light that has sufficient energy to convert thephotosensitizer to an excited state and thereby render it capable ofactivating molecular oxygen to singlet oxygen. The excited state for thephotosensitizer capable of exciting molecular oxygen is generally atriplet state which is more than about 20, usually at least 23 Kcal/molmore energetic than the photosensitizer ground state. Preferably, themedium is irradiated with light having a wavelength of about 450 to 950nm, although shorter wavelengths can be used, for example, 230-950 nm,and longer wavelengths of up to 2000 nm can be used by providingsufficiently intense light to provide for biphotonic excitation.

Although it will usually be preferable to excite the photosensitizer byirradiation with light of a wavelength that is efficiently absorbed bythe photosensitizer, other means of excitation may be used, for example,by energy transfer from an excited state of an energy donor. When anenergy donor is used, wavelengths of light can be used which areinefficiently absorbed by the photosensitizer but are efficientlyabsorbed by the energy donor. The energy donor may be bound to an assaycomponent that is associated, or becomes associated, with thephotosensitizer, for example, bound to a surface or incorporated in theparticle having the photosensitizer. When an energy donor is employedits lowest energy singlet and/or triplet state will usually be of higherenergy than the lowest energy singlet and/or triplet state,respectively, of the photosensitizer.

The singlet oxygen so formed reacts with the photoactive indicatorprecursor to form a photoactive indicator which is fluorescent.Fluorescence of the photoactive indicator that is formed can be detectedfollowing electronic excitation of the photoactive indicator. Normallyelectromagnetic radiation, preferably light, will be used to excite thephotoactive indicator, but energy transfer from molecules that have beenexcited by other means such as chemiexcitation can also be used when thechemiexcitation is separate from the above-mentioned singlet oxygenreaction. The wavelength of light used to excite the photoactiveindicator can be the same or different from the wavelength of light usedto excite the photosensitizer. Usually it will be preferable for thelight emitted by the photoactive indicator to be shorter wavelength thanany fluorescence of the photosensitizer. Preferably, therefore, when thephotosensitizer is fluorescent, the light used to excite the photoactiveindicator will be shorter wavelength than that used to activate thephotosensitizer, usually at least 50 nm shorter, preferably at least 200nm shorter. The fluorescence emitted from the excited photoactiveindicator my be measured in any convenient manner such asphotographically, visually or photometrically, to determine the amountthereof, which is related to the amount of analyte in the medium.

Irradiation of the photosensitizer and the excitation of the photoactiveindicator may be carried out simultaneously but will preferably becarried out sequentially so that the light used to excite thephotosensitizer does not interfere with the fluorescence measurement.The photoactive indicator precursor must not substantially absorb lightat the wavelength used to generate the singlet oxygen and will thereforeusually absorb at shorter wavelengths than the photosensitizer. Inaddition, the photoactive indicator precursor will preferably not absorbsignificantly at the wavelength required to excite the photoactiveindicator and therefore will usually absorb at shorter wavelengths thanthe photoactive indicator.

The method and compositions of the invention may be adapted to mostassays involving sbp members such as ligand-receptor; e.g.,antigen-antibody reactions; polynucleotide binding assays, and so forth.The assays may be homogeneous or heterogeneous, competitive ornoncompetitive. The assay components, photoactive indicator precursorand photosensitizer, can be associated in a number of ways to areceptor, to a ligand, or, when employed, to an surface. The associationmay involve covalent or non-covalent bonds. Those skilled in the artwill be able to choose appropriate associations depending on theparticular assay desired in view of the foregoing and the followingillustrative discussion.

The sample may be pretreated if necessary to remove unwanted materials.The reaction for a noncompetitive sandwich type assay can involve an sbpmember, (e.g., an antibody, polynucleotide probe, receptor or ligand)complementary to the analyte and associated with a photoactive indicatorprecursor; a photosensitizer associated with an sbp member, (e.g.,antibody, polynucleotide probe, receptor or ligand) that is alsocomplementary to the analyte; the sample of interest; and any ancillaryreagents required. In a competitive protocol one sbp member can be aderivative of the analyte and the other sbp member can be complementaryto the analyte, e.g., an antibody. In either protocol the components maybe combined either simultaneously or wholly or partially sequentially.The ability of singlet oxygen produced by an activated photosensitizerto react with the photoactive indicator precursor to form a photoactiveindicator is governed by the binding of an sbp member to the analyte.Hence, the presence or amount of analyte can be determined by measuringthe amount of light emitted upon activation of the photoactive indicatorso formed by irradiation. Both the binding reaction and detection of theextent thereof can be carried out in a homogeneous solution withoutseparation, wherein, preferably, one or both of the photosensitizer andthe photoactive indicator precursor are incorporated in particles towhich the sbp members are attached. This is an advantage of the presentinvention over prior art methods utilizing chemiluminescence.

In a heterogeneous assay approach, one of the sbp members willfrequently be bound to a support or another means provided to separateit from the assay medium. The support may be either a non-dispersiblesurface or a particle. In one embodiment, the support or particle willhave associated with it one member of a group consisting of thephotoactive indicator precursor and the photosensitizer. Another sbpmember will have the other member of the group associated with itwherein the sbp members can independently, either directly orindirectly, bind the analyte or a receptor for the analyte. Thesecomponents are generally combined either simultaneously or wholly orpartially sequentially. The surface or particles are then separated fromthe liquid phase and either are subjected to conditions for activatingthe photosensitizer and the photoactive indicator so formed, usually byirradiating the separated phase, and measuring the amount offluorescence emitted.

Alternatively, a hererogenous assay of this invention may be carried outby providing means such as a surface to separate a first sbp member fromthe liquid assay medium and providing a second sbp member that isassociated with a photosensitizer and that binds to the first sbp memberas a function of the amount of analyte in the medium. The samplesuspected of containing the analyte is then combined with the first andsecond sbp members either simultaneously or wholly or partiallysequentially and the first sbp member is separated from the medium. Athird sbp member associated with a photoactive indicator precursor isthen combined with the separated first sbp member where the third sbpmember is capable of binding directly or indirectly to the second sbpmember. The combination is then irradiated to activate thephotosensitizer and the fluorescence of the photoactive indicator soformed is measured.

The binding reactions in an assay for the analyte will normally becarried out in an aqueous medium at a moderate pH, generally that whichprovides optimum assay sensitivity. Preferably, the activation of thephotosensitizer will also be carried out in an aqueous medium. However,when a separation step is employed, non-aqueous media such as, e.g.,acetonitrile, acetone, toluene, benzonitrile, etc. and aqueous mediawith pH values that are very high, i.e., greater than 10.0, or very low,i.e., less than 4.0, preferably with pH values that are very high, canbe used. As explained above, the assay can be performed either withoutseparation (homogeneous) or with separation (heterogeneous) of any ofthe assay components or products.

The aqueous medium my be solely water or may include from 0.01 to 80volume percent of a cosolvent but will usually include less than 40% ofa cosolvent when an sbp member is used that is a protein. The pH for themedium of the binding reaction will usually be in the range of about 4to 11, more usually in the range of about 5 to 10, and preferably in therange of about 6.5 to 9.5. The pH will usually be a compromise betweenoptimum binding of the binding members and the pH optimum for theproduction of signal and the stability of other reagents of the assay.Usually no change in pH will be required for signal production, althoughif desired, a step involving the addition of an acid or a basic reagentcan be inserted between the binding reaction and generation of singletoxygen and/or signal production. Usually in homogenous assays the finalpH will be in the range of 5-13. For hererogenous assays non-aqueoussolvents may also be used as mentioned above, the main considerationbeing that the solvent not react efficiently with singlet oxygen.

Various buffers may be used to achieve the desired pH and maintain thepH during an assay. Illustrative buffers include borate, phosphate,carbonate, tris, barbital and the like. The particular buffer employedis not critical to this invention, but in an individual assay one oranother buffer may be preferred.

Moderate temperatures are normally employed for carrying out the bindingreactions of proteinaceous ligands and receptors in the assay andusually constant temperature, preferably, 25° to 40°, during the periodof the measurement. Incubation temperatures for the binding reactionwill normally range from about 5° to 45° C., usually from about 15° to40° C., more usually 25° to 40° C. Where binding of nucleic acids occurin the assay, higher temperatures will frequently be used, usually 20°to 90°, more usually 35° to 75° C. Temperatures during measurements,that is, generation of singlet oxygen and light detection, willgenerally range from about 20° to 100°, more usually from about 25° to50° C., more usually 25° to 40° C.

The concentration of analyte which may be assayed will generally varyfrom about 10⁻⁴ to below 10⁻¹⁶ M, more usually from about 10⁻⁶ to 10⁻¹⁴M. Considerations, such as whether the assay is qualitative,semiquantitative or quantitative, the particular detection technique theconcentration of the analyte of interest, and the maximum desiredincubation times will normally determine the concentrations of thevarious reagents.

In competitive assays, while the concentrations of the various reagentsin the assay medium will generally be determined by the concentrationrange of interest of the analyte, the final concentration of each of thereagents will normally be determined empirically to optimize thesensitivity of the assay over the range. That is, a variation inconcentration of the analyte which is of significance should provide anaccurately measurable signal difference.

The concentration of the sbp members will depend on the analyteconcentration, the desired rate of binding, and the degree that the sbpmembers bind nonspecifically. Usually, the sbp members will be presentin at least the lowest expected analyte concentration, preferably atleast the highest analyte concentration expected, and for noncompetitiveassays the concentrations may be 10 to 10⁻⁶ times the highest analyteconcentration but usually less than 10⁻⁴ M, preferably less than 10⁻⁶ M,frequently between 10⁻¹¹ and 10⁻⁷ M. The amount of photosensitizer orphotoactive indicator precursor associated with a sbp member willusually be at least one molecule per sbp member and may be as high as10⁵, usually at least 10-10⁴ when the photosensitizer or photoactiveindicator precursor molecule is incorporated in a particle.

While the order of addition may be varied widely, there will be certainpreferences depending on the nature of the assay. The simplest order ofaddition is to add all the materials simultaneously. Alternatively, thereagents can be combined wholly or partially sequentially. When theassay is competitive, it will often be desirable to add the analyteanalog after combining the sample and an sbp member capable of bindingthe analyte. Optionally, an incubation step may be involved after thereagents are combined, generally ranging from about 30 seconds to 6hours, more usually from about 2 minutes to 1 hour before thephotosensitizer is caused to generate singlet oxygen and the photoactiveindicator is caused to fluoresce.

In a particularly preferred order of addition, a first set of sbpmembers that are complementary to and/or homologous with the analyte arecombined with the analyte followed by the addition of specific bindingpair members complementary to the first specific binding pair members,each associated with a different member of the group consisting of aphotosensitizer and a photoactive indicator precursor. The assaymixture, or a separated component thereof, is irradiated first toproduce singlet oxygen and then later to produce measurablefluorescence.

In a homogeneous assay after all of the reagents have been combined,they can be incubated, if desired. Then, the combination is irradiated(at the necessary wavelengths of light) and the resulting fluorescenceemitted is measured. The emitted fluorescence is related to the amountof the analyte in the sample tested. The amounts of the reagents of theinvention employed in a homogeneous assay depend on the nature of theanalyte. Generally, the homogeneous assay of the present inventionexhibits an increased sensitivity over known assays such as the EMIT®assay. This advantage results primarily because of the improved signalto noise ratio obtained in the present method.

The following assays are provided by way of illustration and notlimitation to enable one skilled in the art to appreciate the scope ofthe present invention and to practice the invention without undueexperimentation. It will be appreciated that the choice of analytes,photosensitizers, photoactive indicator precursors, surfaces, particlesand reaction conditions will be suggested to those skilled in the art inview of the disclosure herein in the examples that follow.

In one embodiment of the invention a photoactive indicator precursor ofthe following formula (Im): ##STR15## is covalently linked to anantibody for human chorionic gonadotropin (HCG) to provide Reagent 1.The photoactive indicator precursor, functionalized with aN-hydroxysuccinimidyl ester of the carboxyl group, reacts with the aminogroups of the antibody. The linking group is a carboxamide. Thephotosensitizer utilized is rose bengal, which is covalently bound tolatex particles having an average dimension of 0.5 micron. The latexparticles and rose bengal are covalently bound to each other by means ofchloromethyl groups on the latex to provide an ester linking group asdescribed in J. Am. Chem. Soc., 97:3741 (1975). The latex particle isfurther linked to a second antibody for HCG by means ofN-hydroxysuccinimidyl ester groups on the latex to provide Reagent 2.Both of the antibodies employed are monoclonal antibodies prepared bystandard hybrid cell line technology. One antibody recognizes theα-subunit of HCG and the other recognizes the β-subunit of HCG. Inconducting the assay a serum sample suspected of containing HCG isobtained from a patient. Fifty microliters of the sample is combined ina 500 microliters of aqueous medium, buffered with Tris buffer at pH8.0, with Reagent 1 and Reagent 2 above. The amounts of Reagent 1 andReagent 2 are sufficient to provide concentrations of each antibody ofabout 10⁻⁹ molar. The reaction mixture is then incubated for a period ofone hour at 25° C. and then irradiated for 30 minutes with 560 nm light.The fluorescence of the solution is then measured by irradiating at 350nm and detecting at 440 nm and is compared with values obtained in asimilar procedure with samples having known concentrations of HCG todetermine the concentration of HCG in the unknown.

In an alternative approach based on the above, Reagent 2 is rose bengalcovalently linked to the second antibody and no latex particle isemployed. In still another alternative approach based on the above,Reagent 2 is rose bengal covalently linked to the second antibody andReagent 1 is the photoactive indicator precursor covalently bound tolatex particles, to which the first antibody is covalently attached. Instill another alternative approach based on the above, Reagent 1 is asdescribed immediately above, Reagent 2 is rose bengal covalently linkedto latex particles, to which avidin is covalently attached, and a thirdreagent (Reagent 2A) that is the second antibody covalently linked tobiotin is employed. Reagent 1 and the third reagent are combined withsample and incubated. Then, an excess of Reagent 2 is added and thereining procedure is as described above.

In another embodiment in accordance with the present invention, a firstset of oil droplets (Reagent 3) is prepared from a solution of thephotosensitizer and chlorophyll in mineral oil in accordance withGiaever, supra. The oil droplets, which range from 0.1 to 2 microns indiameter, are coated with a functionalized surfactant that is linked toa monoclonal antibody for C-reactive protein (CRP). The chlorophyll islipophilic and is therefore dissolved in the lipophilic oil droplet. Asecond set of oil droplets (Reagent 4) is prepared in a similar manner.In this set of droplets the oil droplet is similarly coated with asecond monoclonal antibody for CRP, which recognizes a different portionof the CRP molecule than that recognized by the first monoclonalantibody referred to above. 9-Benzal-10-methyl acridan is irreversiblydissolved in the lipophilic oil droplet by including aN,N-didodecylcarboxamide group bound to one of the phenyl groups of theacridan. The monoclonal antibodies are prepared by standard hybrid cellline technology. A serum sample suspected of containing CRP (50microliters) is combined with excess quantities of Reagent 3 and Reagent4 in an aqueous buffered medium (500 μL) at pH 7.5. The medium isincubated at 25° C. for a period of 20 minutes. The medium, withoutseparation, is irradiated at 633 nm using a HeNs laser for a period oftwenty minutes and the fluorescence of the solution is measured byirradiation at 360 nm and detection at 440 nm of the light emitted. Theintensity of fluorescence is compared with that from samples containingknown amounts of CRP and the amount of CRP in the unknown sample isdetermined by comparing the values. In this way a convenient andsensitive homogeneous immunoassay for CRP is conducted.

In an alternative approach based on the above, Reagent 3 has an antibodyfor fluorescein in place of the antibody for CRP and an additionalreagent (Reagent 3A) has the CRP antibody covalently linked tofluorescein. Reagent 4 has avidin in place of the second CRP antibodyand a fourth reagent (Reagent 4A) has the second antibody covalentlylinked to biotin. In the assay Reagent 4A and Reagent 3A are combinedwith sample and incubated. Thereafter, Reagents 3 and 4 are added andincubated. The remainder of the assay procedure as described above isthen carried out.

In another embodiment of the present invention, one set of liposomes(Reagent 5) (0.2 micron in diameter) is formed by high pressureextrusion of a phospholipid suspension in pH 7.4 buffer through a 0.2micron pore membrane using a commercially available instrument designedfor such purpose. A thyroxine analog is covalently linked to theliposome by first forming mercaptoacetamide groups on the liposome byreaction of phosphatidylethanolamine in the liposome with anN-hydroxysuccinimide ester of methyl carboxymethyl disulfide followed byreaction with dithioerythritol. Bromoacetyl thyroxine is then allowed toreact with the sulfhydrylated liposomes. A metallo-porphyrin dye isdissolved in the lipophilic portion of the liposome. Another set ofliposomes (Reagent 6) is utilized to attach a monoclonal antibody forthyroxine. The antibody is attached covalently by means similar to theattachment of thyroxine. A photoactive indicator precursor of thefollowing formula: ##STR16## is covalently linked by means of acarboxamide linking group to the surface of the liposome. Reagent 5 andReagent 6 are combined in an aqueous buffered assay medium (500 μL) ofpH 8.0 together with a serum sample suspected of containing thyroxinethat contains a thyroxine releasing agent of the following formula:##STR17## to displace thyroxine from binding proteins (50 micro-liters).The assay medium is then incubated at room temperature for 1 hour. Themedium is irradiated at 650 nm for a period of 1 minute and thefluorescence is measured as in the preceding examples. The valueobtained is compared with values obtained by conducting a similar assaywith known amounts of thyroxine. In this way the amount of thyroxine inthe serum sample is quantitated.

In an alternative approach based on the above, Reagent 6 has avidin inplace of antibody for thyroxine. An additional reagent (Reagent 6A) hasantibody for thyroxine covalently linked to biotin. Reagent 5 hasantibody for fluorescein in place of thyroxine and an additional reagent(Reagent 5A) has thyroxine linked covalently to fluorescein. In theassay Reagents 5A and 6A are combined with sample and incubated. Then,Reagent 5 and 6 are added, the mixture is incubated, and the remainderof the assay procedure is followed.

In another embodiment 2-hydroxyethyl-9,10-dibromoanthracene is formedinto a dye crystallite in a manner similar to that described by Gribnau.A 25mer oligonucleotide probe that recognizes a sequence of hepatitis BRNA is covalently attached to the dye crystallite by means of acarbamate linking group. A second 25mer oligonucleotide probe forhepatitis B RNA is covalently linked to 9-(benzal-9H-xanthene) by meansof an amide linking group. The dye crystallite has a particle size 2microns on the average. The oligonucleotides are prepared by standardautomated synthesis technology. A sample (50 μL) from a patientsuspected of having hepatitis B is combined in an aqueous assay medium(500 μL) at pH 7.0 with an excess of the dye crystallite and the secondprobe described above. The assay medium is then incubated at 50° C. fora period of 30 minutes and the fluorescence is then measured byirradiation at 330 nm and detection at 390 nm. The presence of hepatitisB RNA in the sample causes the dye crystallite and latex particles tocome into close proximity by virtue of the binding of the respectiveoligonucleotides with the RNA. Upon irradiation of the medium the9,10-dibromoanthracene is excited and converts ground state oxygen tosinglet oxygen. The singlet oxygen reacts with the 7anthene to give axanthone, which is fluorescent. The fluorescence is measuredphotometrically and the amount of light over a certain threshold levelindicates the presence of hepatitis B RNA in the sample. Irradiation ofthe medium is conducted at room temperature and the assay is conductedin a homogeneous manner to yield an assay for hepatitis B RNA.

In another embodiment the assay is for the determination of a particularblood group antigen on the surface of a red blood cell, namely, an Agroup antiget. Latex particles prepared as described above having aparticle size of 150-500 nm are utilized. The latex particles have anantibody for the A group antigen covalently linked to the latexparticle. The particles also have the photoactive indicator precursor offormula (If): ##STR18## which is dissolved in the latex. This latexparticle reagent is combined in the aqueous medium (500 μl) with wholeblood (100 μl) and 1×10⁻⁴ M of a photosensitizer, which is a hydrophobicdye of the following formula: ##STR19## The hydrophobic dye isincorporated into the red cells present in the sample. The medium isincubated at 25° C. for a period of 10 minutes and then irradiatedat >650 nm with a tungsten source for a period of 30 seconds. Thefluorescence of the solution is then determined by irradiation at 360 nmand detection at 440 nm. The light emitted from the medium is measuredand compared to the amount of light obtained in samples known to be freeof A group antigen red blood cells. Thus, the amount of light over athreshold level indicates the presence of the A blood group antigen.

The present invention further encompasses compositions comprising asuspendible particle of 25 to 4000 nanometer average diameter comprisinga photoactive indicator precursor. The photoactive indicator precursormay be covalently bound to the particle matrix or may be dissolved inthe matrix or dissolved in a solvent that is dissolved in the matrix.The particles will preferably be polymeric or be oil droplets orvesicles such as liposomes. Where the particle is a liposome, thephotoactive indicator precursor will be associated with the lipidbilayer or dissolved in the aqueous interior of the liposome. Theparticle will have an sbp member bound to it. Also encompassed arecompositions comprised of two complementary sbp members bound to eachother wherein one is associated with a photosensitizer and one isassociated with a photoactive indicator precursor.

Another aspect of the present invention relates to kits useful forconveniently performing an assay method of the invention for determiningthe presence or amount of an analyte in a sample suspected of containingthe analyte. To enhance the versatility of the subject invention, thereagents can be provided in packaged combination, in the same orseparate containers,-so that the ratio of the reagents provides forsubstantial optimization of the method and assay. The reagents may eachbe in separate containers or various reagents can be combined in one ormore containers depending on the cross-reactivity and stability of thereagents. The kit comprises (1) a composition wherein the compositioncomprises a suspendible particle comprising a photoactive indicatorprecursor, the particle having an sbp member bound to it, and (2) aphotosensitizer. The photosensitizer can be attached to an sbp member orit can be associated with a particle, to which an sbp member is bound.The kit can further include other separately packaged reagents forconducting an assay including ancillary reagents, and so forth.

Another embodiment of a kit in accordance with the present inventioncomprises in packaged combination a photoactive indicator precursorassociated with a first sbp member and a photosensitizer capable in itsexcited state of activating oxygen to its singlet state associated witha second sbp member.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. Parts and percentages used herein are by weight unlessotherwise specified. Temperatures are in degrees centigrade (°C.). Thefollowing abbreviations are used in the Examples:

"Amino-GATTAG"--a modified 42mer oligonucleotide having the sequenceshown below: ##STR20## with the nucleotide (Clontech Laboratories,#5202-1) at the 5'-end substituted as illustrated below: ##STR21##"Biotin-30mer"--a modified 30mer oligonucleotide having the sequenceshown below: ##STR22## with biotin attached to a modified cytosine(5-methylcytosine) at the 5'-end through a linking group as shown below:##STR23## "CTAATC-30mer"--a modified tailed 30mer oligonucleotide havingthe sequence shown below: ##STR24## "DMF"--dimethyl formamide."EDAC"--1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.

"EDTA"--ethylenediaminetetraacetic acid.

"GATTAG-SH"--a modified 42mer oligonucleotide having the sequence shownbelow: ##STR25## with the 5'-end nucleotide substituted as illustratedbelow: ##STR26## "MES"--2-(N-morpholino)ethane sulfonic acid."SPDP"--N-succinimidyl 3-(2-pyridylthio)-propionate.

"Sulfo-SMCC"--4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

"TCEP"--tris-carboxyethyl phosphine.

"THF"--tetrahydrofuran.

Example 1 Preparation of a Photoactive Indicator Precursor ##STR27##

A solution of coumarin-1 (11.0 g, 47.5 mmol) in ethyl acetate (150 mL)was treated with 10% Pd/C (100 mg) in a parr bottle. The suspension wasthen hydrogenated at 80 psi and 80° C. for 6 hours. The suspension wasfiltered through a bed of celite to remove the Pd/C, and the celite bedwashed with warm ethyl acetate (100 mL). The filtrate was concentratedand dried under vacuo to yield 11.0 g (100%) of the 3,4 -dihydrocoumarin(2) as an oil;

¹ H-NMR (CDCl₃, 250 MHz): δ7.02 (d,J=8.5 Hz,1H); 6.42 (dd,J=8.5 Hz, 1.7Hz, 1H); 6.35 (d,J=1.7 Hz, 1H); 4.07 (q,J=7.0 Hz, 4H); 3.06 (m, 1H);2.80 (dd, J_(gem) =15.6 Hz, J_(vic) =5.4 Hz, 1H); 2.51 (dd, J_(gem)=15.6 Hz, J_(vic) =7.7 Hz, 1H); 1.28 (d,J=7.0 Hz, 3H); 1.15 (t,J=7.0 Hz,6H); MS (EI) calculated for C₁₄ H₁₉ NO₂, 233: found 233 (M⁺, 40%); 218(M⁺ --CH₃, 100%). ##STR28##

A solution of 3,4-dihydrocoumarin 2 (1.68 g, 7.20 mmol) in anhydrous THF(20 mL) was cooled to -78° C. under argon. Lithium diisopropylamide inTHF (8.0 mL of 1.0M, 8.0 mmol) was added to the stirred solution and theresultant yellow colored solution was further stirred for 1 hour. Phenylselenyl chloride (1.50 g, 7.8 mmol) dissolved in THF (10 mL) wassubsequently added into the enolate solution. The orange color of themixture quickly faded to yield a yellow solution. The solution wasstirred for 3 hours and quenched with aqueous NH₄ Cl (10 mL of 1%).After about 10 minutes, dichloromethane (100 mL) was added and theorganic phase separated. The aqueous portion was further extracted withCH₂ Cl₂ (2×20 mL) and the organic portions combined. The combinedorganic portions were washed with brine (20 mL), dried over anhydrousNa₂ SO₄ (25 g) and concentrated to yield 2.10 g of an yellow oil. Theoil was purified by chromatography on silica gel with hexane indichloromethane gradient to yield 1.80 g (67%) of the coumarin-3-phenylselenide 3, as a white powder. Crystallization from hot hexane afforded1.30 g of 3 as white needles, m.p. 99°-101° C.;

¹ H-NMR (C₆ D₆, 250 MHz) δ7.65 (m, 2H); 6.92 (m, 3H); 6.74 (d,J=8.0 Hz,1H); 6.38 (d,J=1.5 Hz, 1H); 6.25 (dd,J=8.0 Hz, 1.5 Hz, 1H); 3.88 (d,J=2Hz, 1H); 3.02 (m, 1H); 2.84 (q,J=7.0 Hz, 4H); 0.96 (d,J=7.0 Hz, 3H);0.80 (t, J=7.0 Hz, 6H); MS (El) calcd. for C₂₀ H₂₃ NO₂ Se 389; found 389(M⁺, 100%); 232 (M⁺ --C₆ H₅ Se 70%); 218 (60%); 202 (45%); UV-Vis(toluene) 300 nm (4600); 310 nm (4600); 330 nm (2100).

Example 2 Preparation of a Photoactive Indicator Precursor ##STR29##

To a stirred suspension of tellurium powder (100 mesh, 13.0 g, 0.10 mol)in dry THF (150 mL) was added a solution of phenyl lithium (60 mL of1.8M, 0.10 mol) in ether-hexanes. The suspension was stirred at roomtemperature for 2 hours and then refluxed for 1 hour. The suspension wasallowed to cool and water (100 mL) was added followed by overnightstirring. Oxygen gas was bubbled through the suspension for 3 hours.Methylene chloride (200 mL) was added and the organic phase separated.The aqueous phase was further extracted with CH₂ Cl₂ (2×100 mL) and thecombined portions washed with brine (100 mL) and dried over anhydrousNa₂ SO₄. The dried solution was passed through a plug of silica (300 g);the filtrate thus obtained was concentrated and crystallized from hotethanol to yield 13.2 g of the diphenyl ditelluride 4, as orange redneedles, m.p. 63-65%C. (lit 63.5°-65° C.); MS (EI) calcd for C₁₂ H₁₀ Te₂414; found 414 (25%); 412 (45%); 410 (50%); 408 (40%); 207 (40%).

The diphenyl ditelluride 4 (1.0 g, 2.5 mmol) was dissolved in TF (10.0mL) and cooled to 0° C. Bromine (125 μL, 2.5 mmol) in THF (5.0 mL) wasadded and the solution stirred at 0° C. for 1 hour and allowed to attainroom temperature. The reaction mixture was stirred at room temperatureuntil no more starting material was detectable by analytical thin layerchromatography to yield compound 5. ##STR30##

A solution of the 3,4-dihydrocoumarin (240 mg, 1.0 mmol) in anhydrousTHF (10 mL) was cooled to -78° C. under argon. Lithium diisopropyl amide(1.1 mL of 1.0M, 1.1 mmol) in THF was added and the solution stirred at-78° C. for 1 hour. A solution of 5 (3 mmol, prepared as describedabove) in THF was cannulated into the ester enolate and the mixturestirred for 2 hours at -78° C. and then allowed to attain roomtemperature. The reaction mixture was quenched with aqueous NH₄ Cl (1%,5 mL) and stirred for another 5 minutes. The reaction mixture was thenextracted with CH₂ Cl₂ (3.25 mL) and the combined organic portionswashed with brine (20 mL) and dried over anhydrous Na₂ SO₄ (20 g).Concentration followed by flash chromatography (under subdued lighting)on silica with CH₂ Cl₂ gave 190 mg (43%) of an yellow oil.Crystallization from cyclohexane afforded 165 mg of the coumarintelluride as a light yellow colored solid;

¹ H-NMR (CDCl₃, 250 MHz) δ7.82 (dd,J=7.0 Hz, 1.2 Hz, 2H); 7.31 (m, 1H);7.26 (m,2H); 6.91 (d,J=8.5 Hz, 1H); 6.38 (dd,J=8.5 Hz, 2.5 Hz, 1H); 6.25(d,J=2.5 Hz, 1H); 4.05 (d, 2.0 Hz, 1H); 3.33 (q,J=7.0 Hz, 4H); 3.25 (m,1H); 1.23 (d,J=7.0 Hz, 3H); 1.16 (t,J=7.0 Hz, 6H); MS (EI) calcd. forC₂₀ H₂₃ NO₂ Te 439 (using ¹³⁰ Te); found 439 (M⁺., 20%); 232 (M⁺ -C₆ H₅Te, 100%); 217 (25%); 202 (35%); UV-Vis (toluene) 310 nm (3860); 330 nm(2400); 370 nm (510).

Example 3 Preparation of a Photoactive Indicator Precursor ##STR31##

A solution of p-bromo-N,N-dimethyl aniline (10.0 g, 50.0 mmol) inanhydrous TF (200 mL) was cooled to -78° C. under argon. Into thiscooled solution was carefully added t-butyl lithium (56 mL of 1.8M, 100mmol) in pentane, and the resulting yellow suspension stirred for 1 hourat -78° C. Finely ground tellurium powder (6.50 g, 50 mmol) was addedunder a stream of argon. The reaction mixture was then allowed to attainroom temperature, by that time (˜2 hours) most of the tellurium haddissolved. The reaction mixture was quenched with water (20 mL) andpoured into aqueous K₃ [Fe(CN)₆ ] solution (17 g in 200 mL, 0.052 mol).The mixture was stirred for 1 hour and then extracted with CH₂ Cl₂(3×200 mL). The combined organic portions were washed with brine (100mL) and dried over anhydrous Na₂ SO₄ (100 g). The dried material waspassed through a plug of silica (300 g) and the filtrate concentrated toyield 12.2 g of an orange-red paste. Crystallization from ethanol gave8.6 g of the ditelluride 7, as an orange red powder. Another batch (2.2g) was recovered from the mother liquor; MS (EI) calculated for C₁₆ H₂₀N₂ Te₂, 500; found 500 (20%); 498 (40%); 496 (45%); 250 (100%); 240(98%).

The ditelluride 7 (1.70 g, 3.4 mmol) was dissolved in a minimum amountof anhydrous THF and cooled to 0° C. The solution was treated withbromine (175 μL, 3.4 mmol) and the mixture stirred at 0° C. for 3 hoursto yield a solution containing the desired product. ##STR32##

The product 8 was then cannulated under argon into a solution of thedihydro coumarin 2 (800 mg, 3.4 mmol) and lithium diisopropyl amide (3.5mL of 1.0M, 3.5 mmol) in THF. The resulting orange red mixture wasallowed to attain room temperature and quenched with aqueous NH₄ Cl (10mL of 1.0%). The mixture was subsequently extracted with CH₂ Cl₂ (3×50mL) and the pooled organic portion dried with brine (50 mL) andanhydrous Na₂ SO₄. Concentration followed by flash chromatography (undersubdued light) on silica with CH₂ Cl₂ gave 510 mg of the coumarin3-(4-dimethylamino)phenyl telluride 9, together with 110 mg of thestarting dihydro coumarin 2. The yield of 9 was 37% based upon recoveredstarting material;

¹ H-NMR (CDCl₃, 250 MHz) δ7.65 (d,J=8.0 Hz, 2H); 6.92 (d,J=8.5 Hz, 1H);6.52 (d,J=8.0 Hz, 2H); 6.41 (dd,J=8.5 Hz, 1.5 Hz, 1H); 6.24 (d,J=1.5 Hz,1H); 4.03 (d, 2 Hz, 1H); 3.38 (q,J=7.0 Hz, 4H); 3.25 (m,H); 2.95 (s,6H);1.19 (d,J=7.0 Hz, 3H); 1.14 (t,J=7.0 Hz, 6H).; MS (EI) calcd. for C₂₂H₂₈ N₂ O₂ Te, 482 (using ¹³⁰ Te); found 482 (M⁺., 20%); 252 (20%); 232(M⁺ --C₈ H₁₀ Te, 100%); UV-Vis (toluene) 300 nm (18000); 320 nm (13600);330 nm (8400).

Example 4 Preparation of Photoactive Indicator Precursor Particles(Acceptor Beads)

A 0.3M solution of coumarin-3-(4-dimethylamino)phenyl telluride 9 wasprepared in degassed ethoxy ethanol by gentle warming. Ethylene glycol(1 mL) was heated to 105°-110° C. in a 4 mL vial. A stock latexsuspension (200 μL of 10% solids in H₂ O) was added to the vial and themixture stirred magnetically under argon.Comarin-3-(4-dimethylamino)phenyl telluride 9 (200 μL, 0.3M inethoxyethanol) was added slowly to the mixture and the resulting mixturestirred for 5 minutes, then allowed to attain room temperature underargon. After cooling, the suspeneion was treated with ethanol (3 mL) andtransferred to a centrifuge tube. The mixture was then centrifuged at15,000 rpm (Sorval, SA 600 rotor) for 1 hour. The supernatant wascarefully decanted and the pellet resuspended in aqueous ethanol (4.0mL) by sonication. The suspension was centrifuged at 15,000 rpm for 1hour. The supernatant was once again removed and the pellet wasresuspended in water (4 mL). Following a final centrifugation andremoval of supernatant, the pellet was resuspended in water to a finalvolume of 2 mL to a yield of 10 mg/mL photoactive indicator precursorparticles suspension.

Example 5 Preparation of Streptavidin-Photoactive Indicator PrecursorDyed Particles

The photoactive indicator precursor particles (1 mL of 10 mg/mL)suspension prepared in Example 4 above was added to an EDAC solution(0.5 mg/mL, 1 mL of 0.02M phosphate buffer, pH 6.0) cooled to 0° C. Thesuspension was stirred under argon for 30 minutes. After this time, thesuspension was added dropwise into a streptavidin solution (5 mg/mL, 1mL) in borate buffer (0.2M, pH 9.0) kept at ˜0° C. The suspension wasstirred for 1 hour and allowed to warmup to room temperature. Water (1mL) was added and the mixture centrifuged at 15,000 rpm for 1 hour. Thesupernatant was discarded and the pellet suspended in water (4 mL) bysonication. The sample was recentrifuged in water (4 mL) by sonication,and after a final centrifugation at 15,000 rpm for 30 minutes, theresultant pellet was suspended in water (5 mL). This gave a 2 mg/mLsuspension of streptavidin-photoactive indicator precursor particles.The presence of streptavidin was confirmed by ³ H biotin binding andquantitated to 2500±250 streptavidin/particle.

Example 6 Preparation of Maleimidated Dextran Photosensitizer Particles

A. Staining of particles.

A dye mixture of chlorophyll-a (2.0 mM) and tetrabutyl squarate (4.0 mM)in benzyl alcohol was prepared. Ethylene glycol (80 mL) was placed in a125 mL Erlenmeyer flask and warmed to 125° C. on a laboratory hot plate.The dye mixture in benzyl alcohol (8 mL) was then added followedimmediately by stock latex suspension (10 mL of 10% solids). Heating wasdiscontinued and the flask and its contents allowed to attain roomtemperature. After cooling, the mixture was diluted with an equal volumeof ethanol and immediately centrifuged at 15,000 rpm for two hours. Thebluish-green supernatant was discarded and the pellet suspended in 50 mLof ethanol by sonication. The suspension was centrifuged at 15,000 rpmfor one hour and the faintly blue supernatant decanted. The pellet wasresuspended in 50% aqueous ethanol (50 mL) by sonication to disperse theparticles. Centrifugation was repeated at 15,000 rpm for an hour. Thesupernatant was decanted and the pellet resuspended in water bysonication. Following a final centrifugation, the pellets wereresuspended in water to a final volume of 20 mL.

B. Preparation of Maleimidated Dextran Photosensitizer Particles.

Aminodextran (500 mg) was partially maleimidated by reacting it withsulfo-SMCC (157 mg, 10 mL H₂ O). The sulf-SMCC was added to a solutionof the aminodextran (in 40 mL, 0.05M Na₂ HPO₄, pH 7.5) and the resultingmixture was incubated for 1.5 hr. The reaction mixture was then dialyzedagainst MES/NaCl (2×2L, 10 mM MES, 10 mM NaCl, pH 6.0, 4° C.). Themaleimidated dextran was centrifuged at 15,000 rpm for 15 minutes andthe supernatant collected. The supernatant dextran solution (54 mL) wasthen treated with imidazole (7 mL of 1.0M solution) in MES buffer (pH6.0) and into this stirred solution was added the stainedphotosensitizer particles (10 mL of 10mg/mL). After stirring for 10minutes the suspension was treated with EDAC (7 mmol in 10 mM pH 6.0MES) and the suspension stirred for 30 minutes. After this time,SurfactAmps® (Pierce) Tween-20 (10%, 0.780 mL) was added to the reactionmixture for a final concentration of 0.1%. The particles were thencentrifuged at 15,000 rpm for 45 minutes and the supernatant discarded.The pellet was resuspended in MES/NaCl (pH 6.0, 10 mM, 100 mL) bysonication. Centrifugation at 15,000 rpm for 45 minutes, followed bypellet resuspension after discarding the supernatant, was performedtwice. The maleimidated dextran photosensitizer particles were stored inwater as a 10 mg/mL suspension.

Example 7 Preparation of GATTAG-Photosensitizer Particles.

Amino-GATTAG (180 μL, 50 mmol) (prepared as described below in Example8) in water was treated with 0.25M borax (50 μL) to give a pH of 9.2.SPDP (50 mg/mL in dry DMF) was added in four aliquots at 0, 10, 20 and30 minutes (33.8 μmol total). The reaction mixture was allowed to standfor 2 hours. Ice cold ethanol (2.1 mL) was added and the product left inthe freezer overnight. The cloudy product mixture was split into twoEppendorf tubes and centrifuged at maximum speed for 10 minutes. Thesupernatant was carefully removed and the pellet dissolved in 400 μL H₂O. Into this solution was added 2.5 M acetate buffer (20 μL, 2.5M, pH5.3).

TCEP in distilled water (10 μL, 20 mM) was added and the reductionallowed to proceed for 30 minutes at room temperature. Absolute ethanol(1.2 mL) was added and the reaction mixture put in the freezer for 2hours. The reaction mixture was centrifuged at full speed in the coldroom and the precipitated GATTAG-SH oligonucleotide was removed as apellet. The pellet was dissolved in 200 μL of 50 mM Na₂ HPO₄ buffer (pH6.85) containing 20 mM EDTA. The solution was degassed and kept underargon. This solution was then added to the maleimidated dextranphotosensitizer particles (14.2 mg/1.5 mL) (prepared above in Example 6)and the reaction mixture allowed to stand overnight. The mixture wascentrifuged at 15,000 rpm for 1 hour and the supernatant discarded. Thepellet was resuspended in water (2 mL) and centrifuged at 15,000 rpm for1 hour. The supernatant was discarded and the pellet resuspended inwater (2 mL). After a final centrifugation the GATTAG-photosensitizerparticles were stored in 2 mL of water solution as a suspension.

Example 8 Assay for Detecting DNA

A. The target 65mer oligonucleotide with the sequence shown below:##STR33## and CTAATC-30mer and amino-CATTAG were prepared on a MilligetBiosearch DNA synthesizer (Model #8750) using standard solid phasephosphoramidite methodology (see Oligonucleotide Symtheses--A PracticalApproach (1984), Gait M. J., Ed., IRL Press Oxford.) The protocolbriefly consisted of (a) removal with dichloroactic acid of the5'-dimethoxytrityl group on the nucleoside attached to the solidsupport; (b) coupling of the incoming nucleoside, which contains a5'-hydroxyl protecting group (preferably dimethoxytrityl) and a3'-hydroxyl protecting group (preferably N,N-diisopropylphosphoramidite), using tetrazole as the catalyst; (c) acapping step with acetic anhydride; and (d) iodine oxidation to convertthe phosphite triester into a phosphate triester. At the conclusion ofthe synthesis ammonium hydroxide was used to (a) cleave the synthesizedpolynucleotide from the support; (b) remove the phosphoryl protectinggroups (β-cyanoethyl); and (c) to remove the base protecting groups. Theoligonucleotide was finally purified by HPLC. B. Biotin-30mer wasprepared similarly as above except that the base of the last incomingnucleotide was a 5-methylcytosine with a protected amine-modifier(American Bionetics, #ABN2599) as shown below: ##STR34##

After deprotection, the free amine was reacted with biotin-LC-NHS(Pierce, #21335G) at a 1:60 molar ratio of the two reagents in 0.1MNaHCO₃, pH 9.0. Following incubation overnight at room temp, theresulting oligonucleotide was analyzed and purified on a 12% denaturingpolyacrylamide gel. C. The assay was performed by mixing various volumes(0-80L of 21 nM) of target 65mer oligonucleotide with CTAATC-30mer (200Lof 15 nM) and biotin-30mer (200 μL of 15 nM) in TRIS/EDTA/NaCl solution(pH 8.0, 100 mM, 0.1 mM, 0.30M, respectively) contained in a 1.5 mLEppendorf tube. The volumes were made up to 0.5 mL and the solutionannealed at 55° C. for 30 minutes so that the 30mer probes couldhybridize with their complements on the target 65mer oligonucleotideupon cooling. The reaction mixture was cooled to room temperature andthen treated with the streptavidin-photoactive indicator precursorparticles (100 μL of 100 μg/mL) followed by GATTAG-photosensitizerparticles (400 μL of 100 μg/mL). The mixture was gently vortexed andallowed to incubate for 2 hours at room temperature. The suspension wasthen transferred to a 12×75 mm test tube and irradiated for 5 minuteswith a Dolan-Jenner lamp (tungsten) equipped with a 610 nm cutofffilter. The sample was treated with an equal volume (1 mL) of buffer andtransferred to a fluorometer. The fluorescence units corresponding to anexcitation with a 360 NB filter and 420 NB filter emission wererecorded. The resulting standard curves for two individual assays areshown in FIG. 1.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes or modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATTAGGATTAGGATTAGGATTAGGATTAGGATTAGGATTAG42                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAATACAGGTTGTTGCCTTCACGCTCGAAA30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGCCGGTGCGCCATGCTCGCCCGCTTCACCTAATCCTAATCCTAATCCTAATCCTAATC60                CTAATC66                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATTAGGATTAGGATTAGGATTAGGATTAGGATTAGGATTAG42                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTGAAGCGGGCGAGCATGGCGCACCGGCAGAGCATTTTCGAGCGTGAAGGCAACAACCTG60                TATTG65                                                                       __________________________________________________________________________

What is claimed is:
 1. A compound of the following formula: ##STR35##wherein R is an organic or organometallic group bound to X through anunsaturated carbon atom, a silicon atom, or a tin atom; and R¹ ishydrogen or alkyl; andX is selenium or tellurium; andwherein theremaining hydrogen atom on the carbon adjacent to the carbon to which XRis bound may be replaced by alkyl or alkylene substituents.
 2. Thecompound of claim 1 which is selected from the group consisting of thefollowing compounds: ##STR36##
 3. A compound of the formula: ##STR37##4. A method for preparing a photoactive indicator, which methodcomprises reacting a compound of the structure: ##STR38## wherein H iscis to the XR group;X is a selenium or tellurium; R is an organic ororganometallic group bound to X through an unsaturated carbon atom, asilicon atom, or a tin atom; and A, when taken with the carbon-carbongroup, forms an alicyclic ring optionally fused to one or more aromaticrings or a heterocyclic ring;with singlet oxygen to yield a photoactiveindicator having an extinction coefficient of at least 10,000 M⁻¹ cm⁻¹at its absorption maximum and a fluorescence emission quantum yield ofat least 0.1.